scholarly journals Cellular invasion into matrix beads: localization of beta 1 integrins and fibronectin to the invadopodia

1991 ◽  
Vol 99 (2) ◽  
pp. 213-225 ◽  
Author(s):  
S.C. Mueller ◽  
W.T. Chen
Keyword(s):  
Cell Surface ◽  
Early Stage ◽  
Matrix Material ◽  
Transformed Cells ◽  
Thin Sections ◽  
Rous Sarcoma ◽  
The Matrix ◽  
Intracellular Compartments ◽  
Beta 1 Integrin ◽  
Cell Invasiveness

We have examined the contribution of adhesion mechanisms to cell invasiveness by growing chicken embryo fibroblasts (CEF) or Rous sarcoma virus-transformed cells (RSVCEF) on fibronectin-coated crosslinked gelatin beads (FN-beads). RSVCEF attached more readily and spread more rapidly on FN-beads than CEF, suggesting an increase in the adhesion-related motility of the transformed cells. In addition, RSVCEF invaded the FN-beads, but CEF did not, by extending specialized cell surface protrusions called invadopodia at sites of cell invasion. FN removal by RSVCEF cultured on prelabeled fluorescent FN-beads (FL-FN) was evident at sites of invadopodia, and internalized FL-FN occurred in vacuoles near the ventral membrane of cells at sites of FN removal. The precise distribution of FN and integrins in cells invading FN-beads was determined by immunofluorescence and immunoelectron microscopy of frozen thin-sections. In both CEF and RSVCEF, beta 1 integrins and FN occupied separate intracellular compartments during the early stage of spreading on FN-beads. Later, beta 1 integrins were largely localized at the ventral cell surface of both CEF and RSVCEF. Polyclonal anti-integrin antibody recognizing beta 1 and several alpha chains, however, labeled both ventral and dorsal cell surfaces. During invasion by RSVCEF, beta 1 integrins were concentrated at extended invadopodia and also colocalized with internalized FL-FN material in phagocytic vesicles. Furthermore, secreted FN was deposited by RSVCEF at the base of invadopodia colocalizing with beta 1 integrin. Both FL-FN matrix removal and formation of the invadopodia were found to be resistant to treatment with GRGDS at concentrations that inhibit the interaction between cells and FN-beads. Thus, the localization of beta 1 integrins to the plasma membrane contacting immobilized FN results in an extremely tight cellular adherence to the matrix bead, that stabilizes invadopodia and also mediates endocytic clearance of degraded FN-matrix material.

scholarly journals AN ELECTRON MICROSCOPE STUDY OF THE FINE STRUCTURE OF FEATHER KERATIN

1962 ◽  
Vol 13 (1) ◽  
pp. 1-12 ◽  
Author(s):  
B. K. Filshie ◽  
G. E. Rogers
Keyword(s):  
Fine Structure ◽  
Electron Microscope ◽  
Matrix Material ◽  
X Ray Diffraction ◽  
Thin Sections ◽  
Apparent Lack ◽  
Feather Keratin ◽  
Fibrous Protein ◽  
The Matrix ◽  
Lead Hydroxide

Thin sections of the rachis of regenerating follicles of pigmented fowl feathers and of mature non-pigmented seagull feather rachis, embedded in methacrylate and Araldite respectively, were studied in the electron microscope. The late stages of development of keratin fibrils were examined in OsO4-fixed follicle material, and after poststaining with lead hydroxide the keratin aggregates were found to be composed of fine microfibrils approximately 30 A in diameter apparently embedded in a matrix material which had absorbed the lead stain. The centre-to-centre separation of the microfibrils was of the order of 35 A. After bulk treatment by reduction with thioglycollic acid, OsO4 staining, and poststaining with lead hydroxide, a similar microfibrillar fine structure was observed in mature rachis. Only after lead staining could the microfibrils be delineated, and their diameter and separation were similar to that found in the keratin of the follicle. It is suggested that feather keratin resembles α-keratins in consisting of microfibrils embedded in an amorphous protein matrix. However, in comparison with α-keratins, the microfibrils are much smaller in diameter, their arrangement is less orderly, and on the basis of the reactions towards the electron staining procedures, the cystine content of the matrix appears to be not greatly different from that of the microfibrils. The significance of a microfibrillar constitution of feather keratin is discussed in relation to current structural models for this fibrous protein deduced from x-ray diffraction studies. The boundaries between the component cells of feather rachis are desmosomal in character and similar to those of related keratinous structures and a number of different types of cells; the melanin granules are dissimilar to those of mammalian epidermis in their apparent lack of melanin-protein lamellae.

scholarly journals Dynamic cytoskeleton-integrin associations induced by cell binding to immobilized fibronectin.

1989 ◽  
Vol 109 (6) ◽  
pp. 3455-3464 ◽  
Author(s):  
S C Mueller ◽  
T Kelly ◽  
M Z Dai ◽  
H N Dai ◽  
W T Chen
Keyword(s):  
Binding Activity ◽  
Integrin Subunit ◽  
Cell Binding ◽  
Con A ◽  
Src Tyrosine Kinase ◽  
Thin Sections ◽  
Rous Sarcoma ◽  
Cellular Attachment ◽  
Sarcoma Virus ◽  
Beta 1 Integrin

We have examined the early events of cellular attachment and spreading (10-30 min) by allowing chick embryonic fibroblasts transformed by Rous sarcoma virus to interact with fibronectin immobilized on matrix beads. The binding activity of cells to fibronectin beads was sensitive to both the mAb JG22E and the GRGDS peptide, which inhibit the interaction between integrin and fibronectin. The precise distribution of cytoskeleton components and integrin was determined by immunocytochemistry of frozen thin sections. In suspended cells, the distribution of talin was diffuse in the cytoplasm and integrin was localized at the cell surface. Within 10 min after binding of cells and fibronectin beads at 22 degrees C or 37 degrees C, integrin and talin aggregated at the membrane adjacent to the site of bead attachment. In addition, an internal pool of integrin-positive vesicles accumulated. The mAb ES238 directed against the extracellular domain of the avian beta 1 integrin subunit, when coupled to beads, also induced the aggregation of talin at the membrane, whereas ES186 directed against the intracellular domain of the beta 1 integrin subunit did not. Cells attached and spread on Con A beads, but neither integrin nor talin aggregated at the membrane. After 30 min, when many of the cells were at a more advanced stage of spreading around beads or phagocytosing beads, alpha-actinin and actin, but not vinculin, form distinctive aggregates at sites along membranes associated with either fibronectin or Con A beads. Normal cells also rapidly formed aggregates of integrin and talin after binding to immobilized fibronectin in a manner that was similar to the transformed cells, suggesting that the aggregation process is not dependent upon activity of the pp60v-src tyrosine kinase. Thus, the binding of cells to immobilized fibronectin caused integrin-talin coaggregation at the sites of membrane-ECM contact, which can initiate the cytoskeletal events necessary for cell adhesion and spreading.

scholarly journals Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells

1981 ◽  
Vol 88 (2) ◽  
pp. 352-357 ◽  
Author(s):  
EG Hayman ◽  
E Engvall ◽  
E Ruoslahti
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Cell Attachment ◽  
Culture Media ◽  
Rat Kidney ◽  
Cell Types ◽  
Transformed Cells ◽  
Kidney Cells ◽  
Normal Cells ◽  
The Matrix

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.

scholarly journals Early-Stage Damage Detection in Advanced Multifunctional Aerospace Composites Using Embedded Carbon Nanotubes and Flocked Carbon Fibers

Proceedings ◽  
2018 ◽  
Vol 2 (8) ◽  
pp. 490
Author(s):  
Latha Nataraj ◽  
Michael Coatney ◽  
Asha Hall ◽  
Mulugeta Haile ◽  
Riley Sherman ◽  
...  
Keyword(s):  
Carbon Nanotubes ◽  
Damage Detection ◽  
Carbon Fibers ◽  
Early Stage ◽  
Matrix Material ◽  
Three Dimensional ◽  
Conductive Network ◽  
The Matrix ◽  
And Performance ◽  
Damage Tolerant

Early-stage damage detection could provide better reliability and performance and a longer lifetime of materials while reducing maintenance time of a variety of structures and systems. We investigate the early-stage damage formation and damage evolution in advanced multi-functional laminated aerospace composites embedded with a very small amount of carbon nanotubes (CNTs) in the matrix material and short carbon fibers along the Z-direction to reinforce the interlaminar interfaces. The three-dimensional (3-D) conductive network formed by the CNTs and the flocked carbon fibers allows for sensitive in-situ damage detection in materials in addition to providing improved mechanical properties such as superior fracture toughness for damage tolerance. We optimize several parameters such as fiber length, diameter, and density to generate an effective 3-D electrical conductive network, and characterize the responses of these composites under mechanical loading to investigate damage formation and evolution, advancing science and technology towards superior damage-tolerant and zero-maintenance structural materials.

scholarly journals Regulation of fibronectin receptor distribution by transformation, exogenous fibronectin, and synthetic peptides.

1986 ◽  
Vol 103 (5) ◽  
pp. 1649-1661 ◽  
Author(s):  
W T Chen ◽  
J Wang ◽  
T Hasegawa ◽  
S S Yamada ◽  
K M Yamada
Keyword(s):  
Cell Surface ◽  
Synthetic Peptide ◽  
Transformed Cells ◽  
Temperature Sensitive ◽  
Fibronectin Receptor ◽  
Receptor Complexes ◽  
Rous Sarcoma ◽  
Cell Surface Structures ◽  
Double Label ◽  
Adhesive Interactions

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 pp60src, indicating that the morphological reversion could occur even in the continued presence of RSV transformation. The synthetic peptide Gly-Arg-Gly-Asp-Ser derived from the sequence of the cell-binding 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.

Determination of the phase structure of polymerblends by electron microscopy

1978 ◽  
Vol 36 (1) ◽  
pp. 492-493
Author(s):  
Dr. G. Kaemof
Keyword(s):  
Screw Extruder ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Single Screw Extruder ◽  
Transmission Electron ◽  
The Matrix ◽  
Twin Screw ◽  
Special Case ◽  
Microscopic Investigations

A mixture of polycarbonate (PC) and styrene-acrylonitrile-copolymer (SAN) represents a very good example for the efficiency of electron microscopic investigations concerning the determination of optimum production procedures for high grade product properties.The following parameters have been varied:components of charge (PC : SAN 50 : 50, 60 : 40, 70 : 30), kind of compounding machine (single screw extruder, twin screw extruder, discontinuous kneader), mass-temperature (lowest and highest possible temperature).The transmission electron microscopic investigations (TEM) were carried out on ultra thin sections, the PC-phase of which was selectively etched by triethylamine.The phase transition (matrix to disperse phase) does not occur - as might be expected - at a PC to SAN ratio of 50 : 50, but at a ratio of 65 : 35. Our results show that the matrix is preferably formed by the components with the lower melting viscosity (in this special case SAN), even at concentrations of less than 50 %.

Polishing process effect on the image of dispersed precipitates

1984 ◽  
Vol 42 ◽  
pp. 534-535
Author(s):  
C.T. Hu ◽  
C.W. Allen
Keyword(s):  
Matrix Material ◽  
Specimen Preparation ◽  
Second Phase ◽  
Precipitate Particle ◽  
Polishing Process ◽  
Second Phase Particles ◽  
The Matrix ◽  
Surface Profiles ◽  
Thinning Process

One important problem in determination of precipitate particle size is the effect of preferential thinning during TEM specimen preparation. Figure 1a schematically represents the original polydispersed Ni3Al precipitates in the Ni rich matrix. The three possible type surface profiles of TEM specimens, which result after electrolytic thinning process are illustrated in Figure 1b. c. & d. These various surface profiles could be produced by using different polishing electrolytes and conditions (i.e. temperature and electric current). The matrix-preferential-etching process causes the matrix material to be attacked much more rapidly than the second phase particles. Figure 1b indicated the result. The nonpreferential and precipitate-preferential-etching results are shown in Figures 1c and 1d respectively.

Application of cross correlation to HREM images of surfaces

1987 ◽  
Vol 45 ◽  
pp. 754-755
Author(s):  
D. E. Luzzi ◽  
L. D. Marks ◽  
M. I. Buckett
Keyword(s):  
Cross Correlation ◽  
A Priori ◽  
Matrix Material ◽  
Correlation Method ◽  
Accurate Knowledge ◽  
Complex Image ◽  
Fourier Filtering ◽  
The Matrix ◽  
Low Pass ◽  
Data Reduction Technique

As the HREM becomes increasingly used for the study of dynamic localized phenomena, the development of techniques to recover the desired information from a real image is important. Often, the important features are not strongly scattering in comparison to the matrix material in addition to being masked by statistical and amorphous noise. The desired information will usually involve the accurate knowledge of the position and intensity of the contrast. In order to decipher the desired information from a complex image, cross-correlation (xcf) techniques can be utilized. Unlike other image processing methods which rely on data massaging (e.g. high/low pass filtering or Fourier filtering), the cross-correlation method is a rigorous data reduction technique with no a priori assumptions.We have examined basic cross-correlation procedures using images of discrete gaussian peaks and have developed an iterative procedure to greatly enhance the capabilities of these techniques when the contrast from the peaks overlap.

Quantification of cell-surface expressed antigenic sites with 5-nm gold markers: Getting close to biologically relevant data

1989 ◽  
Vol 47 ◽  
pp. 1050-1051
Author(s):  
Etienne de Harven ◽  
Hilary Christensen ◽  
Richard Leung ◽  
Cameron Ackerley
Keyword(s):  
Cell Surface ◽  
Human Peripheral Blood ◽  
Contour Length ◽  
Thin Sections ◽  
Gold Particles ◽  
Biologically Relevant ◽  
Transmission Electron ◽  
Entire Cell ◽  
Electron Imaging ◽  
Cell Contour

The T-derived subset of human peripheral blood normal lymphocytes has been selected as a model system to study the usefulness of 5 nm gold markers for quantification of single epitopes expressed on cell surfaces. The chosen epitopes are parts of the CD3 and CD5 molecules and can be specifically identified by hybridoma produced monoclonal antibodies (MoAbs; LEU-4 and LEU-1; Becton-Dick- inson, Mountain view, CA) . An indirect immunolabeling procedure, with goat anti-murine IgG adsorbed on the surface of 5 nm colloidal gold particles (GAM-G5, Janssen Pharmaceutica, Beerse, Belgium) has been used. Backscattered Electron Imaging (BEI) in a field emission scanning electronmicroscope (SEM) and transmission electron microscopy of thin sections of lymphocytes labeled before plastic embedding, were both used to identify and quantitate gold labeled cell surface sites, Estimating that the thickness of “silver” sections is approximately 60 nm and counting the number of gold particles on the entire cell perimeter, we calculated that, for LEU-4, the number of markers per um2 of cell surface is in the 140-160 range (Fig.l). Cell contour length measurements indicated that the surface of one lymphocyte is approximately 130-160 um2 that of a smooth sphere of identical diameter, reflecting the role of microvilli in expanding the surface area. The total number of gold labeled sites on the surface of one lymphocyte averages, therefore between 20,000 and 24,000 per cell.

Cell surface alteration in Epstein-Barr virus-transformed cells from patients with extreme insulin resistance

Diabetes ◽  
1990 ◽  
Vol 39 (8) ◽  
pp. 924-927 ◽  
Author(s):  
D. L. Gorden ◽  
A. Robert ◽  
V. Y. Moncada ◽  
S. I. Taylor ◽  
J. Muhlhauser ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Cell Surface ◽  
Epstein Barr Virus ◽  
Transformed Cells ◽  
Barr Virus ◽  
Surface Alteration ◽  
Extreme Insulin Resistance ◽  
Epstein Barr
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