scholarly journals Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix.

1982 ◽  
Vol 92 (2) ◽  
pp. 485-492 ◽  
Author(s):  
J A McDonald ◽  
D G Kelley ◽  
T J Broekelmann
Keyword(s):  
Extracellular Matrix ◽  
Binding Domain ◽  
Collagen Deposition ◽  
Domain Specific ◽  
Collagen Organization ◽  
Cell Layers ◽  
Fibrillar Component ◽  
Fibronectin Deposition

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti-60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.

MATRIX – In silico and in vitro Models of Angiogenesis: unravelling the role of the extracellular matrix – ERC

Impact ◽  
2017 ◽  
Vol 2017 (4) ◽  
pp. 45-47
Author(s):  
Hans Van Ooosterwyck
Keyword(s):  
Extracellular Matrix ◽  
In Silico ◽  
In Vitro Models

scholarly journals The phosphatase Shp1 interacts with and dephosphorylates cortactin to inhibit invadopodia function

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Alessia Varone ◽  
Chiara Amoruso ◽  
Marcello Monti ◽  
Manpreet Patheja ◽  
Adelaide Greco ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Tyrosine Phosphatase ◽  
Melanoma Cells ◽  
Matrix Degradation ◽  
Extracellular Matrix Degradation ◽  
Invadopodia Formation ◽  
Interference Rna

Abstract Background Invadopodia are actin-based cell-membrane protrusions associated with the extracellular matrix degradation accompanying cancer invasion. The elucidation of the molecular mechanisms leading to invadopodia formation and activity is central for the prevention of tumor spreading and growth. Protein tyrosine kinases such as Src are known to regulate invadopodia assembly, little is however known on the role of protein tyrosine phosphatases in this process. Among these enzymes, we have selected the tyrosine phosphatase Shp1 to investigate its potential role in invadopodia assembly, due to its involvement in cancer development. Methods Co-immunoprecipitation and immunofluorescence studies were employed to identify novel substrate/s of Shp1AQ controlling invadopodia activity. The phosphorylation level of cortactin, the Shp1 substrate identified in this study, was assessed by immunoprecipitation, in vitro phosphatase and western blot assays. Short interference RNA and a catalytically-dead mutant of Shp1 expressed in A375MM melanoma cells were used to evaluate the role of the specific Shp1-mediated dephosphorylation of cortactin. The anti-invasive proprieties of glycerophosphoinositol, that directly binds and regulates Shp1, were investigated by extracellular matrix degradation assays and in vivo mouse model of metastasis. Results The data show that Shp1 was recruited to invadopodia and promoted the dephosphorylation of cortactin at tyrosine 421, leading to an attenuated capacity of melanoma cancer cells to degrade the extracellular matrix. Controls included the use of short interference RNA and catalytically-dead mutant that prevented the dephosphorylation of cortactin and hence the decrease the extracellular matrix degradation by melanoma cells. In addition, the phosphoinositide metabolite glycerophosphoinositol facilitated the localization of Shp1 at invadopodia hence promoting cortactin dephosphorylation. This impaired invadopodia function and tumor dissemination both in vitro and in an in vivo model of melanomas. Conclusion The main finding here reported is that cortactin is a specific substrate of the tyrosine phosphatase Shp1 and that its phosphorylation/dephosphorylation affects invadopodia formation and, as a consequence, the ability of melanoma cells to invade the extracellular matrix. Shp1 can thus be considered as a regulator of melanoma cell invasiveness and a potential target for antimetastatic drugs.

scholarly journals LTBP1 promotes fibrillin incorporation into the extracellular matrix

2021 ◽  
Author(s):  
Matthias Przyklenk ◽  
Veronika Georgieva ◽  
Fabian Metzen ◽  
Sebastian Mostert ◽  
Birgit Kobbe ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Wide Spectrum ◽  
Extracellular Matrix Protein ◽  
Connective Tissues ◽  
Pathogenic Variants ◽  
Human Pathology ◽  
Fibrillin 1

LTBP1 is a large extracellular matrix protein and an associated ligand of fibrillin-microfibrils. Knowledge of LTBP1 functions is largely limited to its role in targeting and sequestering TGFβ growth factors within the extracellular matrix, thereby regulating their bioavailability. However, the recent description of a wide spectrum of phenotypes in multiple tissues in patients harboring LTBP1 pathogenic variants suggests a multifaceted role of the protein in the homeostasis of connective tissues. To better understand the human pathology caused by LTBP1 deficiency it is important to investigate its functional role in extracellular matrix formation. In this study, we show that LTBP1 coordinates the incorporation of fibrillin-1 and -2 into the extracellular matrix in vitro. We also demonstrate that this function is differentially exerted by the two isoforms, the short and long forms of LTBP1. Thereby our findings uncover a novel TGFβ-independent LTBP1 function potentially contributing to the development of connective tissue disorders.

The extracellular matrix of the developing cornea: diversity, deposition and function*

Development ◽  
1988 ◽  
Vol 103 (Supplement) ◽  
pp. 195-205
Author(s):  
J. B. L. Bard ◽  
M. K. Bansal ◽  
A. S. A. Ross
Keyword(s):  
Extracellular Matrix ◽  
Chondroitin Sulphate ◽  
Corneal Stroma ◽  
Experimental System ◽  
Collagen I ◽  
And Function ◽  
20 Nm

This paper examines the role of the extracellular matrix (ECM) in the development of the cornea. After a brief summary of the corneal structure and ECM, we describe evidence suggesting that the differentiation of neural crest (NC) cells into endothelium and fibroblasts is under the control of ocular ECM. We then examine the role of collagen I in stromal morphogenesis by comparing normal corneas with those of homozygous Movl3 mice which do not make collagen I. We report that, in spite of this absence, the cellular morphology of the Movl3 eye is indistinguishable from that of the wild type. In the 16-day mutant stroma, however, the remaining collagens form small amounts of disorganized, thin fibrils rather than orthogonally organized 20 nm-diameter fibrils; a result implying that collagen I plays only a structural role and that its absence is not compensated for. It also suggests that, because these remaining collagens will not form the normal fibrils that they will in vitro, fibrillogenesis in the corneal stroma differs from that elsewhere. The latter part of the paper describes our current work on chick stromal deposition using corneal epithelia isolated with an intact basal lamina that lay down in vitro ∼3μm-thick stromas of organized fibrils similar to that seen in vivo. This experimental system has yielded two unexpected results. First, the amount of collagen and proteoglycans produced by such epithelia is not dependent on whether its substratum is collagenous and we therefore conclude that stromal production by the intact epithelium is more autonomous than hitherto thought. Second, chondroitin sulphate (CS), the predominant proteoglycan, appears to play no role in stromal morphogenesis: epithelia cultured in testicular hyaluronidase, which degrades CS, lay down stromas whose organization and fibrildiameter distribution are indistinguishable from controls. One possible role for CS, however, is as a lubricant which facilitates corneal growth: it could allow fibrils to move over one another without deforming their orthogonal organization. Finally, we have examined the processes of fibrillogenesis in the corneal stroma and conclude that they are different from those elsewhere in the embryo and in vitro, perhaps because there is in the primary stroma an unidentified, highly hydrated ECM macromolecule that embeds the fibrils and that may mediate their morphogenesis.

Membrane changes in neural target cells studied with fluorescent lectin probes

Development ◽  
1983 ◽  
Vol 77 (1) ◽  
pp. 183-200
Author(s):  
L. Gualandris ◽  
P. Rougé ◽  
A. M. Duprat
Keyword(s):  
Extracellular Matrix ◽  
Neural Induction ◽  
Internal Surface ◽  
Target Cells ◽  
Molecular Configuration ◽  
Lectin Receptors ◽  
Molecular Arrangement ◽  
Cell Layers ◽  
Pleurodeles Waltl

The competent ectoderm of Pleurodeles waltl comprises two cell layers with characteristic differences in their morphology, their composition and the molecular arrangement of the various constituents. The use of labelled lectin probes for observations of ectoderm tissue in vitro with u.v. microscopy (epi-illumination) and the quantification of the results show the following:- 1) Differences in labelling according to the nature of the lectins (SB A, PSA, LCA and Con A). These differences provide information on the nature of the carbohydrates which are present at this stage and on the number of receptors. 2) Differences in fluorescence intensity of the surfaces studied. The internal surface of the ectoderm is labelled more densely than the external surface. 3) Rearrangement of the lectin receptors with a new molecular configuration, stressing the fluidity of the membrane (by the mobility of the receptors throughout the membrane) and its importance for the occurrence of neural induction. 4) Existence of membrane glycoconjugate turnover. 5) A difference in behavioural characteristics between the internal and the external surfaces with respect to the lectins and the formation of an extracellular matrix on the internal surface alone. The extracellular matrix seems to have a role in morphogenetic movements.

scholarly journals Tendon Extracellular Matrix Remodeling and Defective Cell Polarization in the Presence of Collagen VI Mutations

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 409 ◽  
Author(s):  
Manuela Antoniel ◽  
Francesco Traina ◽  
Luciano Merlini ◽  
Davide Andrenacci ◽  
Domenico Tigani ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Critical Role ◽  
Active Form ◽  
Congenital Muscular Dystrophy ◽  
Cell Polarization ◽  
Pcr Analysis ◽  
Matrix Remodeling ◽  
Collagen Vi

Mutations in collagen VI genes cause two major clinical myopathies, Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), and the rarer myosclerosis myopathy. In addition to congenital muscle weakness, patients affected by collagen VI-related myopathies show axial and proximal joint contractures, and distal joint hypermobility, which suggest the involvement of tendon function. To gain further insight into the role of collagen VI in human tendon structure and function, we performed ultrastructural, biochemical, and RT-PCR analysis on tendon biopsies and on cell cultures derived from two patients affected with BM and UCMD. In vitro studies revealed striking alterations in the collagen VI network, associated with disruption of the collagen VI-NG2 (Collagen VI-neural/glial antigen 2) axis and defects in cell polarization and migration. The organization of extracellular matrix (ECM) components, as regards collagens I and XII, was also affected, along with an increase in the active form of metalloproteinase 2 (MMP2). In agreement with the in vitro alterations, tendon biopsies from collagen VI-related myopathy patients displayed striking changes in collagen fibril morphology and cell death. These data point to a critical role of collagen VI in tendon matrix organization and cell behavior. The remodeling of the tendon matrix may contribute to the muscle dysfunction observed in BM and UCMD patients.

Role of galaptin in ovarian carcinoma adhesion to extracellular matrix in vitro

1990 ◽  
Vol 43 (1) ◽  
pp. 43-57 ◽  
Author(s):  
Howard J. Allen ◽  
Daniel Sucato ◽  
Barbara Woynarowska ◽  
Sally Gottstine ◽  
Ashu Sharma ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Ovarian Carcinoma

Three-Dimensional Simulation of In Vitro Angiogenesis: Effects of Extracellular Matrix Structure and Density

2010 ◽  
Author(s):  
Lowell Taylor Edgar ◽  
James E. Guilkey ◽  
Clayton J. Underwood ◽  
Brenda Baggett ◽  
Urs Utzinger ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Three Dimensional ◽  
Vascular Endothelial ◽  
Chemical Factors ◽  
Dimensional Simulation ◽  
Endothelial Growth Factor ◽  
In Vitro Angiogenesis

The process of angiogenesis is regulated by both chemical and mechanical signaling. While the role of chemical factors such as vascular endothelial growth factor (VEGF) during angiogenesis has been extensively studied, the influence of the mechanostructural environment on new vessel generation has received significantly less attention. During angiogenesis, endothelial cells in the existing vasculature detach and migrate out into the surrounding extracellular matrix (ECM), forming tubular structures that eventually mature into new blood vessels. This process is modulated by the structure and composition of the ECM [1]. The ECM is then remodeled by endothelial cells in the elongating neovessel tip, resulting in matrix condensation and changes in fiber orientation [2]. The mechanism as to how angiogenic vasculature and the ECM influence each other is poorly understood.

scholarly journals Mutations in the Gal83 Glycogen-Binding Domain Activate the Snf1/Gal83 Kinase Pathway by a Glycogen-Independent Mechanism

2004 ◽  
Vol 24 (1) ◽  
pp. 352-361 ◽  
Author(s):  
Heather A. Wiatrowski ◽  
Bryce J. W. van Denderen ◽  
Cristin D. Berkey ◽  
Bruce E. Kemp ◽  
David Stapleton ◽  
...  
Keyword(s):  
Glycogen Synthase ◽  
Metabolic Stress ◽  
Binding Domain ◽  
Glycogen Accumulation ◽  
Snf1 Kinase ◽  
Transcriptional Regulatory ◽  
Amp Activated Protein Kinase

ABSTRACT The yeast Snf1 kinase and its mammalian ortholog, AMP-activated protein kinase (AMPK), regulate responses to metabolic stress. Previous studies identified a glycogen-binding domain in the AMPK β1 subunit, and the sequence is conserved in the Snf1 kinase β subunits Gal83 and Sip2. Here we use genetic analysis to assess the role of this domain in vivo. Alteration of Gal83 at residues that are important for glycogen binding of AMPK β1 abolished glycogen binding in vitro and caused diverse phenotypes in vivo. Various Snf1/Gal83-dependent processes were upregulated, including glycogen accumulation, expression of RNAs encoding glycogen synthase, haploid invasive growth, the transcriptional activator function of Sip4, and activation of the carbon source-responsive promoter element. Moreover, the glycogen-binding domain mutations conferred transcriptional regulatory phenotypes even in the absence of glycogen, as determined by analysis of a mutant strain lacking glycogen synthase. Thus, mutation of the glycogen-binding domain of Gal83 positively affects Snf1/Gal83 kinase function by a mechanism that is independent of glycogen binding.

Single-chain antibody fragments derived from a human synthetic phage-display library bind thrombospondin and inhibit sickle cell adhesion

Blood ◽  
2003 ◽  
Vol 102 (2) ◽  
pp. 718-724 ◽  
Author(s):  
Nicholas A. Watkins ◽  
Lily M. Du ◽  
J. Paul Scott ◽  
Willem H. Ouwehand ◽  
Cheryl A. Hillery
Keyword(s):  
Sickle Cell ◽  
Matrix Protein ◽  
Binding Domain ◽  
Extracellular Matrix Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Adhesion Assay ◽  
Single Chain ◽  
Cell Binding

AbstractThe enhanced adhesion of sickle red blood cells (RBCs) to the vascular endothelium and subendothelial matrix likely plays a significant role in the pathogenesis of vaso-occlusion in sickle cell disease. Sickle RBCs have enhanced adhesion to the plasma and extracellular matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. In this study, we sought to develop antibodies that bind TSP from a highly diverse library of human single-chain Fv fragments (scFvs) displayed on filamentous phage. Following 3 rounds of phage selection of increasing stringency 6 unique scFvs that bound purified TSP by enzyme-linked immunosorbent assay were isolated. Using an in vitro flow adhesion assay, 3 of the 6 isolated scFvs inhibited the adhesion of sickle RBCs to immobilized TSP by more than 40% compared with control scFvs (P < .001). Furthermore, scFv TSP-A10 partially inhibited sickle RBC adhesion to activated endothelial cells (P < .005). Using TSP proteolytic fragments to map the binding site, we showed that 2 of the inhibitory scFvs bound an epitope in the calcium-binding domain or proximal cell-binding domain of TSP, providing evidence for the role of these domains in the adhesion of sickle RBCs to TSP. In summary, we have isolated a panel of scFvs that specifically bind to TSP and differentially inhibit sickle RBC adhesion to surface-bound TSP under flow conditions. These scFvs will be useful reagents for investigating the role of the calcium and cell-binding domains of TSP in sickle RBC adhesion.

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