scholarly journals Cell-to-substrate contacts in an adhesion-defective mutant of Balb/c3T3 cells

1981 ◽  
Vol 52 (1) ◽  
pp. 183-196
Author(s):  
J.R. Yates ◽  
C.S. Izzard
Keyword(s):  
Cell Surface ◽  
Close Contact ◽  
Cell Types ◽  
Separation Distance ◽  
Specific Substrate ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Defective Mutant ◽  
Mutant Cells ◽  
Cell Thickness

The cell-to-substrate contacts of the adhesion-defective mutant, AD6, have been examined by interference reflexion microscopy and compared with those of the wild-type Balb/c3T3 cell. The 2 cell-types differed in their ability to produce focal contacts with the substrate (10-15 nm separation distance). Only 10% of AD6 versus 92% of Balb/c3T3 cells formed these contacts. When present in AD6 the focal contacts were smaller and fewer in number per cell than in Balb/c3T3. Close contact with the substrate (approx. 30 nm separation distance) was formed by both cell types. The absence of the stronger focal contacts accounts for the reduced substrate adhesion of the mutant cells and for other phenotypic characteristics such as reduced spreading, rounded shape, altered pattern of movement, and absence of stress fibres. The reduced adhesion of the mutant has been attributed to a change in the pattern of glycoproteins exposed at the cell surface, which results from a defect in glycosylation. This suggests that normal glycosylation and correct exposure of one or more cell surface components are required for the formation of a specific substrate adhesion, the focal contact, and offers the possibility of identifying individual surface components involved in formation of this adhesion. Attention is drawn to zero-order minima originating from cell thickness and not cell-to-substrate separation distance in the interference reflexion image.

Formation of cell-to-substrate contacts during fibroblast motility: an interference-reflexion study

1980 ◽  
Vol 42 (1) ◽  
pp. 81-116
Author(s):  
C.S. Izzard ◽  
L.R. Lochner
Keyword(s):  
Close Contact ◽  
Leading Edge ◽  
Linear Structure ◽  
Separation Distance ◽  
Primary Role ◽  
Focal Contact ◽  
Focal Contacts ◽  
Spread Area ◽  
Chick Heart ◽  
Planar Substrates

The formatin of close contacts and focal contacts (ca. 30 nm and 10–15 nm separation distance respectively) has been studied during the movement of chick heart fibroblasts on planar substrates using interference-reflexion microscopy, and evaluated in the context of spreading and net movement. During spreading the overall advance of the margin of the close contact is steady, punctuated by periods in which it remains stationary, and only 5% of the time is spent withdrawing in contrast to the extreme leading edge. The close contact advances only where a lamellipodium has first extended free of the substrate (greater than or equal to 100 nm separation distance) ahead of the existing close contact. The new close contact is formed by the lamellipodium lowering to the substrate either progressively from its base forward or distally in patches which later join with the main close contact. New focal contacts are formed successively ahead of existing ones, either by microspikes or lamellipodia contacting the substrate locally ahead of the close contact, or within the close contact usually immediately, but not more than 1–2 microns, behind its margin. Examining the cell margin alternately with interference-reflexion and differential-interference contrast showed that the formation of the focal contact was preceded in 90% of the cases by the development of a linear structure in the form of a microspike (as expected), a short projection (< 2 microns long) of the lamellipodium, or a fibre within the lamellipodium, each of which could be traced to the cytoplasmic fibre typically associated with the focal contact. Stress fibres subsequently developed centripetally from these initial fibres. The different forms of the linear structure which preceded the focal contact were interchangeable, giving rise to one another, and we have evaluated that the structure common to each is probably a short bundle of microfilaments. The following features indicate that the close contact plays a primary role in marginal spreading: it is lost when spreading ceases; it is reformed when spreading resumes but only under the newly spread area; the advance of the margin of the thicker leading lamella closely follows that of the close contact; the advance of both can occur ahead of and is thus independent of existing focal contacts and associated stress fibres. We propose that the close contact provides the adhesion required to transmit to the substrate the forces involved in the forward movements of the marginal cytoplasm. The continual formation of focal contacts and stress fibres at the margin is consistent with their role, suggested by others, in drawing the bulk of the cell forward. These evaluations are discussed in the context of the form and distribution of contractile proteins in the cell margin. A primary role of the lamellipodia and microspikes in extending the cell margin and forming new adhesions, preparatory to further cytoplasmic movement, is established by this work.

scholarly journals Cell surface distribution of fibronectin and vitronectin receptors depends on substrate composition and extracellular matrix accumulation.

1988 ◽  
Vol 106 (6) ◽  
pp. 2171-2182 ◽  
Author(s):  
I I Singer ◽  
S Scott ◽  
D W Kawka ◽  
D M Kazazis ◽  
J Gailit ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Cell Attachment ◽  
Cellular Migration ◽  
Surface Distribution ◽  
Fibronectin Receptor ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Double Label ◽  
Coated Surfaces

We used antibodies against the alpha subunits of the human fibronectin receptor (FNR) and vitronectin receptor (VNR) to localize simultaneously FNR and VNR at major substrate adhesion sites of fibroblasts and melanoma cells with double-label immunofluorescence microscopy. In early (2-6-h) serum-containing cultures, both FNR and VNR coaccumulated in focal contacts detected by interference reflection microscopy. Under higher resolution immunoscanning electron microscopy, FNR and VNR were also observed to be distributed randomly on the dorsal cell surface. As fibronectin-containing extracellular matrix fibers accumulated beneath the cells at 24 h, FNR became concentrated at contacts with these fibers and was no longer detected at focal contacts. VNR was not observed at matrix contacts but remained strikingly localized in focal contacts of the 24-h cells. Since focal contacts represent the sites of strongest cell-to-substrate adhesion, these results suggest that FNR and VNR together play critical roles in the maintenance of stable contacts between the cell and its substrate. In addition, the accumulation of FNR at extracellular matrix contacts implies that this receptor might also function in the process of cellular migration along fibronectin-containing matrix cables. To define the factors governing accumulation of FNR and VNR at focal contacts, fibroblasts in serum-free media were plated on substrates coated with purified ligands. Fibronectin-coated surfaces fostered accumulation of FNR but not VNR at focal contacts. On vitronectin-coated surfaces, or substrata derivatized with a tridecapeptide containing the cell attachment sequence Arg-Gly-Asp, both FNR and VNR became concentrated at focal contacts. These observations suggest that the availability of ligand is critical to the accumulation of FNR and VNR at focal contacts, and that FNR might also recognize substrate-bound vitronectin.

scholarly journals Talin-Null Cells of Dictyostelium Are Strongly Defective in Adhesion to Particle and Substrate Surfaces and Slightly Impaired in Cytokinesis

1997 ◽  
Vol 138 (2) ◽  
pp. 349-361 ◽  
Author(s):  
Jens Niewöhner ◽  
Igor Weber ◽  
Markus Maniak ◽  
Annette Müller-Taubenberger ◽  
Günther Gerisch
Keyword(s):  
Cell Surface ◽  
Gene Replacement ◽  
Cell Cortex ◽  
Mitotic Apparatus ◽  
Small Areas ◽  
Substrate Adhesion ◽  
Substrate Surfaces ◽  
Cell To Cell Adhesion ◽  
Neuronal Growth Cones ◽  
Mutant Cells

Dictyostelium discoideum contains a full-length homologue of talin, a protein implicated in linkage of the actin system to sites of cell-to-substrate adhesion in fibroblasts and neuronal growth cones. Gene replacement eliminated the talin homologue in Dictyostelium and led to defects in phagocytosis and cell-to-substrate interaction of moving cells, two processes dependent on a continuous cross talk between the cell surface and underlying cytoskeleton. The uptake rate of yeast particles was reduced, and only bacteria devoid of the carbohydrate moiety of cell surface lipopolysaccharides were adhesive enough to be recruited by talin-null cells in suspension and phagocytosed. Cell-to-cell adhesion of undeveloped cells was strongly impaired in the absence of talin, in contrast with the cohesion of aggregating cells mediated by the phospholipid-anchored contact site A glycoprotein, which proved to be less talin dependent. The mutant cells were still capable of moving and responding to a chemoattractant, although they attached only loosely to a substrate via small areas of their surface. With their high proportion of binucleated cells, the talin-null mutants revealed interactions of the mitotic apparatus with the cell cortex that were not obvious in mononucleated cells.

Cell-to-substrate contacts in living fibroblasts: an interference reflexion study with an evaluation of the technique

1976 ◽  
Vol 21 (1) ◽  
pp. 129-159 ◽  
Author(s):  
C.S. Izzard ◽  
L.R. Lochner
Keyword(s):  
Interference Pattern ◽  
Close Contact ◽  
Numerical Aperture ◽  
Separation Distance ◽  
Zero Order ◽  
Glass Substrates ◽  
Focal Contacts ◽  
History Of ◽  
Chick Heart ◽  
Peripheral Regions

The closeness of contact between cultured chick heart fibroblasts and glass substrates has been examined by interference reflexion microscopy. Evaluation of the optical technique demonstrated that both the film of medium between the cell and substrate and the cell itself act as two superimposed thin films which generate a complex interference pattern in reflected light or low illuminating numerical aperture (I.N.A.). The interference pattern generated by the cell consists of first and higher orders of interference and can be eliminated from the image by increasing the I.N.A. to greater than 1.0. The residual zero-order interference pattern at high I.N.A. originates from the thin film of medium between cell and substrate and corresponds to the closeness of contact between cell and substrate. Based on the zero-order interference pattern and a comparison of the same cells with differential interference optics, the following features were recognized in living chick heart fibroblasts. Focal contacts, 0.25-0.5 mum wide, 2–10 mum long, separated by 10–15 nm from the substrate, are located under the peripheral regions of the leading lamellae and near the edge of extended non-spreading regions of the cell margin in moving and stationary cells. Individual focal contacts are coincident with cytoplasmic fibres of the same dimensions as the contact or with the peripheral ends of longer fibres that extend from the focal contact toward the centre of the cell. In spreading cells a second type of contact is present under the peripheral regions of the leading lamellae. This close contact consists of a broad area of uniform cell-to-substrate separation distance (ca. 30 nm). Focal contacts are distributed within the close contact under the leading lamellae. Lamellipodia extend ahead of the close contact and are separated by 100 nm or more from the substrate. Depending on the previous motile history of the cell, the close contact extends to varying degrees under the centre of the cell but it is typically dissected here by discrete areas of distinctly greater cell-to-substrate separation (100-140 nm). Evidence for the adhesive nature of the focal contacts is considered. The common identity is discussed of the focal contacts and associated cytoplasmic fibres described here in living cells with the regions of closest apposition to the substrate and associated cytoplasmic plaques and bundles of microfilaments seen in EM studies.

Fibronexus-Like Adhesion Sites are Present at the Invasive Zones of Human Epidermoid Carcinomas

1982 ◽  
Vol 40 ◽  
pp. 106-109
Author(s):  
Irwin I. Singer
Keyword(s):  
Cell Cycle ◽  
Serum Concentration ◽  
Substrate Binding ◽  
High Serum ◽  
Adhesion Sites ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Adhesion Complex ◽  
Low Serum

Our previous results indicate that two types of fibronectin-cytoskeletal associations may be formed at the fibroblast surface: dorsal matrixbinding fibronexuses generated in high serum (5% FBS) cultures, and ventral substrate-adhering units formed in low serum (0.3% FBS) cultures. The substrate-adhering fibronexus consists of at least vinculin (VN) and actin in its cytoplasmic leg, and fibronectin (FN) as one of its major extracellular components. This substrate-adhesion complex is localized in focal contacts, the sites of closest substratum approach visualized with interference reflection microscopy, which appear to be the major points of cell-tosubstrate adhesion. In fibroblasts, the latter substrate-binding complex is characteristic of cultures that are arrested at the G1 phase of the cell cycle due to the low serum concentration in their medium. These arrested fibroblasts are very well spread, flattened, and immobile.

The organization of cell surface membrane domains

1989 ◽  
Vol 47 ◽  
pp. 804-805
Author(s):  
Michael Edidin
Keyword(s):  
Cell Surface ◽  
Membrane Lipids ◽  
Surface Membrane ◽  
Lateral Diffusion ◽  
Cell Types ◽  
Membrane Domains ◽  
Membrane Biology ◽  
Morphological Polarity ◽  
Discrete Domains ◽  
Membrane Components

Cell surface membranes are based on a fluid lipid bilayer and models of the membranes' organization have emphasised the possibilities for lateral motion of membrane lipids and proteins within the bilayer. Two recent trends in cell and membrane biology make us consider ways in which membrane organization works against its inherent fluidity, localizing both lipids and proteins into discrete domains. There is evidence for such domains, even in cells without obvious morphological polarity and organization [Table 1]. Cells that are morphologically polarised, for example epithelial cells, raise the issue of membrane domains in an accute form.The technique of fluorescence photobleaching and recovery, FPR, was developed to measure lateral diffusion of membrane components. It has also proven to be a powerful tool for the analysis of constraints to lateral mobility. FPR resolves several sorts of membrane domains, all on the micrometer scale, in several different cell types.

Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies

1979 ◽  
Author(s):  
S. Korach ◽  
D. Ngo
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Vascular Endothelial Cells ◽  
Intercellular Junctions ◽  
Cell Types ◽  
Endothelial Damage ◽  
Antibody Concentration ◽  
High Yields ◽  
Scanning Em

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.

scholarly journals Cellular Receptors Involved in KSHV Infection

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 118
Author(s):  
Emma van der Meulen ◽  
Meg Anderton ◽  
Melissa J. Blumenthal ◽  
Georgia Schäfer
Keyword(s):  
Virus Infection ◽  
Cell Surface ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Cell Types ◽  
Target Cells ◽  
Specific Cell ◽  
Cellular Receptors ◽  
Diverse Range ◽  
Kshv Infection

The process of Kaposi’s Sarcoma Herpes Virus’ (KSHV) entry into target cells is complex and engages several viral glycoproteins which bind to a large range of host cell surface molecules. Receptors for KSHV include heparan sulphate proteoglycans (HSPGs), several integrins and Eph receptors, cystine/glutamate antiporter (xCT) and Dendritic Cell-Specific Intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). This diverse range of potential binding and entry sites allows KSHV to have a broad cell tropism, and entry into specific cells is dependent on the available receptor repertoire. Several molecules involved in KSHV entry have been well characterized, particularly those postulated to be associated with KSHV-associated pathologies such as Kaposi’s Sarcoma (KS). In this review, KSHV infection of specific cell types pertinent to its pathogenesis will be comprehensively summarized with a focus on the specific cell surface binding and entry receptors KSHV exploits to gain access to a variety of cell types. Gaps in the current literature regarding understanding interactions between KSHV glycoproteins and cellular receptors in virus infection are identified which will lead to the development of virus infection intervention strategies.

Functional partitioning of beta1 integrins revealed by activating and inhibitory mAbs

1997 ◽  
Vol 110 (21) ◽  
pp. 2647-2659 ◽  
Author(s):  
M.T. Cruz ◽  
C.L. Dalgard ◽  
M.J. Ignatius
Keyword(s):  
Cell Surface ◽  
Actin Cytoskeleton ◽  
Dermal Fibroblasts ◽  
The Other ◽  
Embryonic Chick ◽  
Cell Surfaces ◽  
Double Labeling ◽  
Focal Contacts ◽  
Functional Partitioning ◽  
Discrete Populations

Integrins exist in different activation states on the surfaces of cells. Addition of the proper signal, ligand, or antibody can alter the activation state of these molecules. We report here the identification of two immunocytochemically distinct populations of beta1 integrins on fixed embryonic chick dermal fibroblasts. One population, recognized by the integrin activating mAb TASC, localizes to discrete regions of the cell, most likely focal contacts. These integrins co-localize with other proteins, such as vinculin and F-actin, and their retention at these sites is dependent on the actin cytoskeleton. The other population, identified with the inhibitory mAb W1B10, is more evenly distributed throughout the cell surface, and its pattern remains unchanged after disruption of the actin cytoskeleton. Double labeling experiments using Fab fragments of TASC alongside whole W1B10 IgG revealed non-overlapping staining patterns. These results show that it is possible to visualize and study discrete populations of integrins on cell surfaces using two different antibodies. We hypothesize that these antibodies report differences in the distribution of receptors in two different states. A model is proposed describing the ligand independent recruitment of integrins based on these findings and results from other labs.

Intracellular traffic of the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP3 to the apical plasma membrane of MDCK and Caco-2 cells: apical targeting occurs in the absence of N-glycosylation

2000 ◽  
Vol 113 (23) ◽  
pp. 4193-4202 ◽  
Author(s):  
N.R. Meerson ◽  
V. Bello ◽  
J.L. Delaunay ◽  
T.A. Slimane ◽  
D. Delautier ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Transmembrane Proteins ◽  
Cell Types ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Intracellular Traffic ◽  
Transmembrane Glycoprotein ◽  
Direct Demonstration ◽  
Targeting Signal

Glycosylation was considered the major signal candidate for apical targeting of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non-glycosylated apical transmembrane proteins usually do not reach the cell surface. Here we were able to follow the targeting of the apical transmembrane glycoprotein NPP3 both when glycosylated and non-glycosylated. Transfected in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic (35)S-labeling and surface immunoprecipitation. The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surface in Caco-2 cells. In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface. In the presence of tunicamycin, NPP3 was not N-glycosylated. The non-glycosylated protein was partially retained intracellularly but the fraction that reached the cell surface was nevertheless predominantly targeted apically. However, transcytosis of the non-glycosylated protein was partially impaired in MDCK cells. These results provide direct evidence that glycosylation cannot be considered an apical targeting signal for NPP3, although glycosylation is necessary for correct trafficking of the protein to the cell surface.

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