scholarly journals The distribution of distinct integrins in focal contacts is determined by the substratum composition

1989 ◽  
Vol 92 (1) ◽  
pp. 67-75 ◽  
Author(s):  
K.R. Fath ◽  
C.J. Edgell ◽  
K. Burridge
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Culture Conditions ◽  
Fibronectin Receptor ◽  
Vitronectin Receptor ◽  
Normal Culture ◽  
Focal Contacts ◽  
Cytoplasmic Face ◽  
Predominant Form

The distribution of two integrins, the fibronectin receptor and the vitronectin receptor, has been explored in an endothelium-derived cell line plated onto various substrata. On a fibronectin substratum, in the presence of serum, these cells develop focal contacts that contain the fibronectin receptor, whereas the vitronectin receptor is diffusely distributed over the cell surface. Conversely, cells plated onto vitronectin-coated coverslips concentrate only the vitronectin receptor within focal contacts. The accumulation of the vitronectin receptor within focal contacts also occurs when the cells are plated on uncoated coverslips but in the presence of serum. Therefore, we conclude that under normal culture conditions (i.e. in serum-containing media), the vitronectin receptor is the predominant form of integrin involved in substratum adhesion. This conclusion is supported by experiments in which cells were cultured on fibronectin-coated coverslips in the presence of serum. Initially these cells developed focal contacts containing only the fibronectin receptor. Within several hours, however, there was a progressive replacement of focal contacts containing the fibronectin receptor by focal contacts expressing the vitronectin receptor. After approximately 12 h in culture, most cells contained focal contacts expressing only the vitronectin receptor. Focal contacts containing either the fibronectin or vitronectin receptor were both associated with the termini of stress fibres and contained the proteins talin and vinculin. These observations lead us to propose that the cell does not discriminate between these different integrins when assembling the cytoskeletal components at the cytoplasmic face of focal contacts.

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 Integrin Dynamics and Matrix Assembly

2000 ◽  
Vol 148 (5) ◽  
pp. 1075-1090 ◽  
Author(s):  
Roumen Pankov ◽  
Edna Cukierman ◽  
Ben-Zion Katz ◽  
Kazue Matsumoto ◽  
Diane C. Lin ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Matrix Assembly ◽  
Fibronectin Receptor ◽  
Vitronectin Receptor ◽  
Focal Contacts ◽  
Dominant Negative Inhibitor ◽  
Translocation Mechanism ◽  
Cytoskeletal Component ◽  
Receptor Ligation

Fibronectin matrix assembly is a multistep, integrin-dependent process. To investigate the role of integrin dynamics in fibronectin fibrillogenesis, we developed an antibody-chasing technique for simultaneous tracking of two integrin populations by different antibodies. We established that whereas the vitronectin receptor αvβ3 remains within focal contacts, the fibronectin receptor α5β1 translocates from focal contacts into and along extracellular matrix (ECM) contacts. This escalator-like translocation occurs relative to the focal contacts at 6.5 ± 0.7 μm/h and is independent of cell migration. It is induced by ligation of α5β1 integrins and depends on interactions with a functional actin cytoskeleton and vitronectin receptor ligation. During cell spreading, translocation of ligand-occupied α5β1 integrins away from focal contacts and along bundles of actin filaments generates ECM contacts. Tensin is a primary cytoskeletal component of these ECM contacts, and a novel dominant-negative inhibitor of tensin blocked ECM contact formation, integrin translocation, and fibronectin fibrillogenesis without affecting focal contacts. We propose that translocating α5β1 integrins induce initial fibronectin fibrillogenesis by transmitting cytoskeleton-generated tension to extracellular fibronectin molecules. Blocking this integrin translocation by a variety of treatments prevents the formation of ECM contacts and fibronectin fibrillogenesis. These studies identify a localized, directional, integrin translocation mechanism for matrix assembly.

Ultrastructural Study of a differentiating human colon carcinoma cell line

1990 ◽  
Vol 48 (3) ◽  
pp. 208-209
Author(s):  
Sylvie Polak-Charcon ◽  
Mehrdad Hekmati ◽  
Yehuda Ben Shaul
Keyword(s):  
Cell Line ◽  
Morphological Changes ◽  
Secretory Granules ◽  
Human Colon ◽  
Cell Types ◽  
Culture Conditions ◽  
Morphological Evidence ◽  
Human Colon Carcinoma Cell ◽  
Colon Cell

The epithelium of normal human colon mucosa “in vivo” exhibits a gradual pattern of differentiation as undifferentiated stem cells from the base of the crypt of “lieberkuhn” rapidly divide, differentiate and migrate toward the free surface. The major differentiated cell type of the intestine observed are: absorptive cells displaying brush border, goblet cells containing mucous granules, Paneth and endocrine cells containing dense secretory granules. These different cell types are also found in the intestine of the 13-14 week old embryo.We present here morphological evidence showing that HT29, an adenocarcinoma of the human colon cell line, can differentiate into various cell types by changing the growth and culture conditions and mimic morphological changes found during development of the intestine in the human embryo.HT29 cells grown in tissue-culture dishes in DMEM and 10% FCS form at late confluence a multilayer of morphologically undifferentiated cell culture covered with irregular microvilli, and devoid of tight junctions (Figs 1-3).

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.

scholarly journals Human macrophage maturation and heterogeneity: analysis with a newly generated set of monoclonal antibodies to differentiation antigens

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1257-1264 ◽  
Author(s):  
R Andreesen ◽  
KJ Bross ◽  
J Osterholz ◽  
F Emmrich
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Cell Lineage ◽  
Culture Conditions ◽  
Human Macrophage ◽  
Blood Monocytes ◽  
Differentiation Antigens

We have analyzed the expression of late differentiation antigens during terminal in vitro maturation of human macrophages (M phi) from blood monocytes (MO) in comparison to their distribution among mature M phi residing in various tissue sites. By immunizing mice with M phi derived from blood MO by culture on hydrophobic Teflon foils, monoclonal antibodies (mAbs) were developed (MAX.1, MAX.2, MAX.3, MAX.11) that reacted with lineage-restricted differentiation antigens. These antigens were expressed exclusively on M phi or were markedly increased after in vitro differentiation. The only overlap to another hemopoietic cell lineage was observed with MAX.3, which is shared by platelets and megakaryocytes. In the course of M phi maturation in vitro, the MAX.1 and MAX.3 antigens are detected within the cytoplasm two days before they appear on the cell surface. In contrast, the MAX.11 antigen is expressed simultaneously in the cytoplasm and at the cell surface, is found in varying degrees on a minor portion of blood MO and U937 cells, and is expressed rapidly at high density during early M phi differentiation in vitro. Among conventional mAbs that do not react with MO we found those against the transferrin (TF)-receptor, the BA-2, and the PCA1 antigen to label M phi. M phi matured in vivo and isolated from body fluids were positive with some but not all MAX mAbs. Distinctive patterns were observed with pulmonary M phi, exudate M phi from pleural and peritoneal effusions, synovial fluids, and early lactation milk. M phi from the alveolar space, for example, constantly expressed the MAX.2 antigen but not the MAX.3 antigen. Pleural effusion M phi, however, did not react with the MAX.1 mAb, but in most cases, it did react with the MAX.3 mAb. The detection of novel differentiation antigens, all expressed on monocyte-derived M phi but differently expressed on site-specific M phi in situ, underlines the remarkable heterogeneity among human M phi. The expression of these antigens is flexible because those MAX antigens that were not expressed in situ could be induced if cells from distinct tissue sites were cultured in vitro for several days. MAX mAbs may be of potential value to study both the sequential stages of maturation within the M phi lineage as well as differential developments induced by various culture conditions in parallel to environmental factors in vivo.

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