Isolation of laminin-1 and type IV collagen from the EHS sarcoma

1994 ◽  
Vol 16 (3-4) ◽  
pp. 231-233 ◽  
Author(s):  
Hynda K. Kleinman
Keyword(s):  
Type Iv Collagen ◽  
Type Iv ◽  
Laminin 1

scholarly journals Assembly of the exogenous extracellular matrix during basement membrane formation by alveolar epithelial cells in vitro

2000 ◽  
Vol 113 (5) ◽  
pp. 859-868 ◽  
Author(s):  
A. Furuyama ◽  
K. Mochitate
Keyword(s):  
Epithelial Cells ◽  
Basement Membrane ◽  
Type Iv Collagen ◽  
Alveolar Epithelial Cells ◽  
Coarse Mesh ◽  
Lamina Densa ◽  
Alveolar Epithelial ◽  
Type Iv ◽  
Laminin 1

We found that immortalized alveolar type II epithelial cells (SV40-T2 cells) that were cultured on dense fibrillar collagen supplemented with Matrigel gel formed a thin and continuous lamina densa beneath them. Immunohistochemical analysis of laminin-1, type IV collagen, entactin (nidogen) and perlecan in the culture indicated that all these components were integrated into a sheet structure of basement membrane beneath the cells. Analysis of the temporal and spatial distribution of the basement membrane macromolecules revealed that the initial deposits of laminin-1 and entactin were significantly greater in area in the presence of Matrigel. These globular deposits and the coarse mesh of basement membrane macromolecules developed into a flat membranous basement membrane. In the absence of Matrigel, the SV40-T2 cells failed to form a continuous lamina densa, and the deposits stayed in the coarse mesh. The major biotinylated Matrigel components that were integrated into the basement membrane were laminin-1 and entactin. Furthermore, SV40-T2 cells supplemented with exogenous laminin-1 alone as well as laminin-1 contaminated with entactin formed a continuous lamina densa. These results indicate that the laminin-1 and entactin supplied from the Matrigel were incorporated into a basement membrane beneath the SV40-T2 cells, and contributed to the formation of basement membrane. Therefore, we concluded that the alveolar epithelial cells synthesize laminin-1, entactin, type IV collagen, and perlecan, but that they also needed to assemble exogenous laminin-1 into the basement membrane to complete its formation in vitro.

scholarly journals Evidence for the location of a binding sequence for the α2β1 integrin of endothelial cells, in the β1 subunit of laminin

1995 ◽  
Vol 309 (3) ◽  
pp. 765-771 ◽  
Author(s):  
P A Underwood ◽  
F A Bennett ◽  
A Kirkpatrick ◽  
P A Bean ◽  
B A Moss
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Type Iv Collagen ◽  
Collagen Type ◽  
Collagen Type Iv ◽  
Collagen Types ◽  
Type Iv ◽  
Integrin Binding Site ◽  
Beta 1 Integrin ◽  
Laminin 1

To date no specific location on laminin 1 for the binding of alpha 2 beta 1 integrin has been described, although recent evidence supports a location in the E1XNd fragment of the cross region. We have identified a peptide sequence from this region, in the beta 1 chain of laminin 1, YGYYGDALR, which inhibits the adhesion of endothelial cells to laminin 1 and type-IV collagen. A structurally related sequence from the CNBr-cleaved fragment CB3 of the alpha 1 chain of collagen type IV, FYFDLR, inhibits endothelial cell adhesion to both collagen types I and IV and laminin 1. The CB3 fragment containing the FYFDLR sequence has been shown to contain binding sites for both alpha 1 beta 1 and alpha 2 beta 1 integrins. Present experiments with anti-integrin antibodies indicate that the alpha 2 beta 1 integrin on endothelial cells can account for all the cell binding to collagen types I and IV, and that this integrin makes a major contribution towards the adhesion of these cells to laminin 1. We therefore propose that the peptide FYFDLR participates in alpha 2 beta 1 binding to collagen type IV and that the putatively structurally similar peptide, YGYYGDALR, participates in alpha 2 beta 1 binding to laminin 1. This is the first account of structurally related peptide sequences from laminin 1 and type-IV collagen which show reciprocal inhibition of cell adhesion to either ligand and which might form part of a common integrin-binding site, as well as the first suggestion of a precise location contributing to the alpha 2 beta 1 integrin binding site on laminin 1.

scholarly journals Laminin assembles into separate basement membrane and fibrillar matrices in Schwann cells

2002 ◽  
Vol 115 (5) ◽  
pp. 1005-1015 ◽  
Author(s):  
Maria V. Tsiper ◽  
Peter D. Yurchenco
Keyword(s):  
Basement Membrane ◽  
Schwann Cell ◽  
Type Iv Collagen ◽  
Competent Cell ◽  
Β1 Integrin ◽  
Cell Surfaces ◽  
Lamina Densa ◽  
Blocking Antibody ◽  
Type Iv ◽  
Laminin 1

Laminins are important for Schwann cell basement membrane assembly and axonal function. In this study, we found that exogenous laminin-1, like neuromuscular laminins-2/4, formed two distinct extracellular matrices on Schwann cell surfaces, each facilitated by laminin polymerization. Assembly of one, a densely-distributed reticular matrix, was accompanied by a redistribution of cell-surface dystroglycan and cytoskeletal utrophin into matrix-receptor-cytoskeletal complexes. The other, a fibrillar matrix,accumulated in separate zones associated with pre-existing β1-integrin arrays. The laminin-1 fragment E3 (LG-modules 4-5), which binds dystroglycan and heparin, inhibited reticular-matrix formation. By contrast,β1-integrin blocking antibody (Ha2/5) prevented fibrillar assembly. Ultrastructural analysis revealed that laminin treatment induced the formation of a linear electron-dense extracellular matrix (lamina densa)separated from plasma membrane by a narrow lucent zone (lamina lucida). This structure was considerably reduced with non-polymerizing laminin, fully blocked by E3, and unaffected by Ha2/5. Although it formed in the absence of type IV collagen, it was nonetheless able to incorporate this collagen. Finally, cell competency to bind laminin and form a basement membrane was passage-dependent. We postulate that laminin induces the assembly of a basement membrane on competent cell surfaces probably mediated by anchorage through LG 4-5. Upon binding, laminin interacts with dystroglycan,mobilizes utrophin, and assembles a `nascent' basement membrane, independent of integrin, that is completed by incorporation of type IV collagen. However,the fibrillar β1-integrin dependent matrix is unlikely to be precursor to basement membrane.

A Post-Embedding Colloidal Gold Immunocytochemical Approach To The Study Of Matrix Accumulation In Glomerular Basement Membrane.

1999 ◽  
Vol 5 (S2) ◽  
pp. 1336-1337
Author(s):  
Caroline A. Miller ◽  
Dominic Cosgrove
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Type Iv Collagen ◽  
Immunohistochemical Analysis ◽  
Ultrastructural Localization ◽  
Type Iv ◽  
Progressive Loss ◽  
Matrix Accumulation ◽  
Immunocytochemical Approach ◽  
Laminin 1

Alport renal disease pathogenesis is characterized by a progressive irregular thickening, thinning, and splitting of the glomerular basement membrane (GBM), which culminates in a focal and segmental glomerulonephritis and progressive loss of glomerular filtration, leading to uremia and death. A mouse model for this disease was produced using a gene targeting approach (Cosgrove et al., 1996). The resulting model displays renal pathology that is very similar to that observed in humans. As matrix accumulation has long been associated with the thickened regions of the GBM, this model provided a means to study the molecular composition and ultrastructural localization of matrix in these rarefied regions of the GBM in the Alport mouse.We examined three matrix molecules based on preliminary data; type IV collagen α l and α2 chains, laminin-1 and fibronectin. Immunohistochemical analysis showed that while all three of these molecules localize primarily to the mesangial matrix of normal mouse glomeruli, in the Alport glomeruli these molecules seem to be heavily deposited in the GBM.

The use of 1nm silver enhanced gold probes for the detection of skin antigens

1990 ◽  
Vol 48 (3) ◽  
pp. 902-903
Author(s):  
J.P Cassella ◽  
H. Shimizu ◽  
A. Ishida-Yamamoto ◽  
R.A.J. Eady
Keyword(s):  
Type Iv Collagen ◽  
Freeze Substitution ◽  
Collagen Type Iv ◽  
Electron Microscopic ◽  
Normal Human Skin ◽  
Type Iv ◽  
Type Vii Collagen ◽  
Keratin Filaments ◽  
Normal Human ◽  
Phosphate Buffered Saline

1nm colloidal gold with silver enhancement has been used in conjunction with a low-temperature post-embedding (post-E) technique for the demonstration of skin antigens at both the light microscopic (LM) and electron microscopic (EM) levels.Keratin filaments and basement membrane zone (BMZ) associated antigens in normal human skin (NHS) were immunolabelled using antibodies against keratin 14, 10, and 1, the carboxy-terminus and collagenous portion of type VII collagen, type IV collagen and bullous pemphigoid antigen (BP-Ag).Fresh samples of NHS were cryoprotected in 15% glycerol, cryofixed in propane at -190°C, subjected to freeze substitution in methanol at -80°C and embedded in Lowicryl K11M at -60°C. Polymerisation of the resin was initiated under UVR at - 60°C for 48 hours and continued at room temperature for a further 48 hours. Semith in sections were air dried onto slides coated with 3-aminopropyltriethoxysilane. The following immunolabelling protocol was adopted: Primary antibody was applied for 2 hours at 37°C or overnight at 4°C. Following washing in Dulbecco’s phosphate buffered saline (PBSA) a biotinylated secondary antibody was applied for 2 hours at 37°C. The sections were further washed in PBSA and 1nm gold avidin was applied. Sections were finally washed in PBSA and silver enhanced.

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