scholarly journals Fibronectin Regulates Assembly of Actin Filaments and Focal Contacts in Cultured Cells via the Heparin-binding Site in Repeat III13

1999 ◽  
Vol 10 (5) ◽  
pp. 1521-1536 ◽  
Author(s):  
Laird Bloom ◽  
Kenneth C. Ingham ◽  
Richard O. Hynes
Keyword(s):  
Matrix Protein ◽  
Cultured Cells ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Extracellular Matrix Protein ◽  
Heparin Binding ◽  
Actin Organization ◽  
Focal Contacts ◽  
Α5β1 Integrin ◽  
Amino Acid Segment

Fibroblasts, when plated on the extracellular matrix protein fibronectin (FN), rapidly spread and form an organized actin cytoskeleton. This process is known to involve both the central α5β1 integrin-binding and the C-terminal heparin-binding regions of FN. We found that within the heparin-binding region, the information necessary for inducing organization of stress fibers and focal contacts was located in a 29–amino acid segment of FN type III module 13 (III13). We did not find a cytoskeleton-organizing role for repeat III14, which had previously been implicated in this process. Within III13, the same five basic amino acids known to be most important for heparin binding were also necessary for actin organization. A substrate of III13 alone was only weakly adhesive but strongly induced formation of filopodia and lamellipodia. Stress fiber formation required a combination of III13 and III7–11(which contains the integrin α5β1 recognition site), either as a single fusion protein or as separate polypeptides, and the relative amounts of the two binding sites appeared to determine whether stress fibers or filopodia and lamellipodia were the predominant actin structures formed. We propose that a balance of signals from III13 and from integrins regulates the type of actin structures assembled by the cell.

scholarly journals Extracellular matrix fibronectin mediates an endothelial cell response to shear stress via the heparin-binding, matricryptic RWRPK sequence of FNIII1H

2016 ◽  
Vol 311 (4) ◽  
pp. H1063-H1071 ◽  
Author(s):  
William Okech ◽  
Keren M. Abberton ◽  
Julia M. Kuebel ◽  
Denise C. Hocking ◽  
Ingrid H. Sarelius
Keyword(s):  
Extracellular Matrix ◽  
Shear Stress ◽  
Matrix Protein ◽  
Umbilical Vein ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Extracellular Matrix Protein ◽  
Heparin Binding ◽  
Flow Conditions ◽  
Endothelial Cell Response

Endothelial cells (EC) respond to mechanical forces such as shear stress in a variety of ways, one of which is cytoskeletal realignment in the direction of flow. Our earlier studies implicated the extracellular matrix protein fibronectin in mechanosensory signaling to ECs in intact arterioles, via a signaling pathway dependent on the heparin-binding region of the first type III repeat of fibrillar fibronectin (FNIII1H). Here we test the hypothesis that FNIII1H is required for EC stress fiber realignment under flow. Human umbilical vein ECs (HUVECs) exposed to defined flow conditions were used as a well-characterized model of this stress fiber alignment response. Our results directly implicate FNIII1H in realignment of stress fibers in HUVECs and, importantly, show that the matricryptic heparin-binding RWRPK sequence located in FNIII1 is required for the response. Furthermore, we show that flow-mediated stress fiber realignment in ECs adhered via α5β1-integrin-specific ligands does not occur in the absence of FHIII1H, whereas, in contrast, αvβ3-integrin-mediated stress fiber realignment under flow does not require FNIII1H. Our findings thus indicate that there are two separate mechanosignaling pathways mediating the alignment of stress fibers after exposure of ECs to flow, one dependent on αvβ3-integrins and one dependent on FNIII1H. This study strongly supports the conclusion that the RWRPK region of FNIII1H may have broad capability as a mechanosensory signaling site.

Improved in vitro rheological system for studying the effect of fluid shear stress on cultured cells

1993 ◽  
Vol 265 (1) ◽  
pp. C289-C298 ◽  
Author(s):  
H. J. Schnittler ◽  
R. P. Franke ◽  
U. Akbay ◽  
C. Mrowietz ◽  
D. Drenckhahn
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Matrix Protein ◽  
Fluid Shear Stress ◽  
Cultured Cells ◽  
Cellular Adhesion ◽  
Fiber Formation ◽  
Extracellular Matrix Protein ◽  
Fluid Shear

A rheological in vitro system has been developed to study and quantify cellular adhesion under precisely defined external shear forces. The system is similar to a cone-and-plate viscosimeter. A rotating transparent cone produces both steady and pulsatile flow profiles on cultured cells. Direct visualization of cells by phase-contrast or fluorescence optics and connection of the optical system to a computer-controlled x/y-linear stage allows automatic recording of any point of the cell cultures. With the use of up to 12 individual rheological units, this setup allows the quantitative analysis of cell substrate adhesion by determination of cell detachment kinetics. Two examples of application of this rheological system have been studied. First, we show that the extracellular matrix protein laminin strongly increases endothelial cell adhesion under fluid shear stress. In a second approach, we obtained further support for the concept that shear stress-induced formation of actin filament stress fibers is important for endothelial cells to resist the fluid shear stress; inhibition of stress fiber formation by doxorubicin resulted in significant detachment of endothelial cells exposed to medium levels of fluid shear stress (5 dyn/cm2). No detachment was seen under resting conditions.

scholarly journals Identification of the growth factor–binding sequence in the extracellular matrix protein MAGP-1

2020 ◽  
Vol 295 (9) ◽  
pp. 2687-2697 ◽  
Author(s):  
Thomas J. Broekelmann ◽  
Nicholas K. Bodmer ◽  
Robert P. Mecham
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Matrix Protein ◽  
Transforming Growth Factor ◽  
Cultured Cells ◽  
Transforming Growth Factor Β ◽  
Bmp Signaling ◽  
Extracellular Matrix Protein ◽  
Surface Plasmon Resonance Analysis ◽  
Factor Binding

Microfibril-associated glycoprotein-1 (MAGP-1) is a component of vertebrate extracellular matrix (ECM) microfibrils that, together with the fibrillins, contributes to microfibril function. Many of the phenotypes associated with MAGP-1 gene inactivation are consistent with dysregulation of the transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling system. We have previously shown that full-length MAGP-1 binds active TGFβ-1 and some BMPs. The work presented here further defines the growth factor–binding domain of MAGP-1. Using recombinant domains and synthetic peptides, along with surface plasmon resonance analysis to measure the kinetics of the MAGP-1–TGFβ-1 interaction, we localized the TGFβ- and BMP-binding site in MAGP-1 to a 19-amino acid–long, highly acidic sequence near the N terminus. This domain was specific for binding active, but not latent, TGFβ-1. Growth factor activity experiments revealed that TGFβ-1 retains signaling activity when complexed with MAGP-1. Furthermore, when bound to fibrillin, MAGP-1 retained the ability to interact with TGFβ-1, and active TGFβ-1 did not bind fibrillin in the absence of MAGP-1. The absence of MAGP was sufficient to raise the amount of total TGFβ stored in the ECM of cultured cells, suggesting that the MAGPs compete with the TGFβ large latent complex for binding to microfibrils. Together, these results indicate that MAGP-1 plays an active role in TGFβ signaling in the ECM.

scholarly journals Structure and binding properties of collagen type XIV isolated from human placenta.

1993 ◽  
Vol 120 (2) ◽  
pp. 557-567 ◽  
Author(s):  
J C Brown ◽  
K Mann ◽  
H Wiedemann ◽  
R Timpl
Keyword(s):  
Human Placenta ◽  
Matrix Protein ◽  
Cell Attachment ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Extracellular Matrix Protein ◽  
Neutral Salt ◽  
Heparin Binding ◽  
Collagen Vi ◽  
Sulphate Proteoglycan

Collagen XIV was isolated from neutral salt extracts of human placenta and purified by several chromatographic steps including affinity binding to heparin. The same procedures also led to the purification of a tissue form of fibronectin. Collagen XIV was demonstrated by partial sequence analysis of its Col1 and Col2 domains and by electron microscopy to be a disulphide-linked molecule with a characteristic cross-shape. The individual chains had a size of approximately 210 kD, which was reduced to approximately 180 kD (domain NC3) after treatment with bacterial collagenase. Specific antibodies mainly to NC3 epitopes were obtained by affinity chromatography and used in tissue and cell analyses by immunoblotting and radioimmunoassays. Two sequences from NC3 were identified on fragments obtained after trypsin cleavage. They were identical to cDNA-derived sequences of undulin, a noncollagenous extracellular matrix protein. This suggests that collagen XIV and undulin may be different splice variants from the same gene. Heparin binding was confirmed in ligand assays with a large basement membrane heparan sulphate proteoglycan. This binding could be inhibited by heparin and heparan sulphate but not by chondroitin sulphate. In addition, collagen XIV bound to the triple helical domain of collagen VI. The interactions with heparin sulphate proteoglycan and collagen VI were not shared by the NC3 domain, or by reduced and alkylated collagen XIV. No or only low binding was observed for collagens I-V, pN-collagens I and III, and several noncollagenous matrix proteins, including laminin, recombinant nidogen, BM-40/osteonectin, plasma and tissue fibronectin, vitronectin, and von Willebrand factor. Insignificant activity was also shown in cell attachment assays with nine established cell lines.

Characterization of Cardiac Function and Arterial Mechanics During Early Postnatal Development in Fibulin-5 Null Mice

2013 ◽  
Author(s):  
Victoria Le ◽  
Hiromi Yanagisawa ◽  
Jessica Wagenseil
Keyword(s):  
Matrix Protein ◽  
Elastic Fiber ◽  
Connective Tissue Disorder ◽  
Fiber Formation ◽  
Extracellular Matrix Protein ◽  
Elastic Fibers ◽  
Arterial Mechanics ◽  
Early Postnatal Development ◽  
Complex Process

Fibulin-5 is an extracellular matrix protein that interacts with other proteins during a complex process that results in elastic fiber formation from the elastin precursor, tropoelastin [1]. Elastic fibers are an important component of tissues requiring elasticity, including large arteries, lungs and skin. In mice lacking fibulin-5 ( Fbln5−/−), these tissues contain disorganized elastic fibers and exhibit decreased elasticity [2]. The phenotype of Fbln5−/− mice is similar to that of humans with cutis laxa, a connective tissue disorder characterized by loose skin and narrow arteries with reduced compliance.

Macromolecular specializations that mediate lateral interactions between microfilaments and the membrane at focal contacts

1992 ◽  
Vol 50 (1) ◽  
pp. 724-725
Author(s):  
Steven J. Samuelsson ◽  
Paul W. Luther ◽  
David W. Pumplin ◽  
Robert J. Bloch
Keyword(s):  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Focal Contact ◽  
Lateral Interactions ◽  
Matrix Assembly ◽  
Cytoplasmic Surface ◽  
Focal Contacts ◽  
Specific Proteins

Focal contacts are membrane specializations of cultured cells where stress fibers terminate and where the cell is most closely applied to the substrate. The organization of this cytoskeletal-membrane-extracellular matrix assembly has been well characterized. Immunofluorescence microscopy has shown that two focal contact-specific proteins, vinculin and talin, colocalize with microfilaments for several microns before the stress fiber terminates. This result raises the question of whether microfilament-membrane interactions are limited to the ends of microfilaments, or if lateral interactions also occur. We addressed this question by examining the cytoplasmic surface of isolated focal contacts in detail.

scholarly journals Clinically-identified C-terminal mutations in fibulin-3 are prone to misfolding and destabilization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
DaNae R. Woodard ◽  
Emi Nakahara ◽  
John D. Hulleman
Keyword(s):  
Matrix Protein ◽  
Cultured Cells ◽  
Open Angle Glaucoma ◽  
Culture Conditions ◽  
Glycosylation Site ◽  
Age Related Macular Degeneration ◽  
Extracellular Matrix Protein ◽  
Macular Dystrophy ◽  
Alternative Mechanism ◽  
Age Related

AbstractDistinct mutations in the secreted extracellular matrix protein, fibulin-3 (F3), have been associated with a number of ocular diseases ranging from primary open angle glaucoma to cuticular age-related macular degeneration to a rare macular dystrophy, Malattia Leventinese (ML). The R345W F3 mutation that causes ML leads to F3 misfolding, inefficient secretion and accumulation at higher intracellular steady state levels in cultured cells. Herein, we determined whether fifteen other clinically-identified F3 mutations also led to similar levels of misfolding and secretion defects, which might provide insight into their potential pathogenicity. Surprisingly, we found that only a single F3 variant, L451F, presented with a significant secretion defect (69.5 ± 2.4% of wild-type (WT) F3 levels) and a corresponding increase in intracellular levels (226.8 ± 25.4% of WT F3 levels). Upon follow-up studies, when this conserved residue (L451) was mutated to a charged (Asp or Arg) or bulky (Pro, Trp, Tyr) residue, F3 secretion was also compromised, indicating the importance of small side chains (Leu, Ala, or Gly) at this residue. To uncover potential inherent F3 instability not easily observed under typical culture conditions, we genetically eliminated the sole stabilizing N-linked glycosylation site (N249) from select clinically-identified F3 mutants. This removal exacerbated R345W and L451F secretion defects (19.8 ± 3.0% and 12.4 ± 1.2% of WT F3 levels, respectively), but also revealed a previously undiscovered secretion defect in another C-terminal variant, Y397H (42.0 ± 10.1% of WT F3 levels). Yet, glycan removal did not change the relative secretion of the N-terminal mutants tested (D49A, R140W, I220F). These results highlight the uniqueness and molecular similarities between the R345W and L451F variants and also suggest that previously identified disease-associated mutations (e.g., R140W) are indistinguishable from WT with respect to secretion, hinting that they may lead to disease by an alternative mechanism.

Transmembrane CEACAM1 affects integrin-dependent signaling and regulates extracellular matrix protein–specific morphology and migration of endothelial cells

Blood ◽  
2005 ◽  
Vol 105 (10) ◽  
pp. 3925-3934 ◽  
Author(s):  
Mario M. Müller ◽  
Bernhard B. Singer ◽  
Esther Klaile ◽  
Björn Öbrink ◽  
Lothar Lucka
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Focal Adhesion ◽  
Matrix Protein ◽  
Network Formation ◽  
Adhesion Formation ◽  
Fiber Formation ◽  
Extracellular Matrix Protein ◽  
And Migration ◽  
Laminin 1

AbstractCarcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1/CD66a), expressed on leukocytes, epithelia, and endothelia mediates homophilic cell adhesion. It plays an important role in cell morphogenesis and, recently, soluble CEACAM1 isoforms have been implicated in angiogenesis. In the present study, we investigated the function of long transmembrane isoform of CEACAM1 (CEACAM1-L) in cultured rat brain endothelial cells. We observed that expression of CEACAM1-L promotes network formation on basement membrane Matrigel and increased cell motility after monolayer injury. During cell-matrix adhesion, CEACAM1-L translocated into the Triton X-100–insoluble cytoskeletal fraction and affected cell spreading and cell morphology on Matrigel and laminin-1 but not on fibronectin. On laminin-1, CEACAM1-L–expressing cells developed protrusions with lamellipodia, showed less stress fiber formation, reduced focal adhesion kinase (FAK) tyrosine phosphorylation, and decreased focal adhesion formation leading to high motility. CEACAM1-L–mediated morphologic alterations were sensitive to RhoA activation via lysophosphatidic acid (LPA) treatment and dependent on Rac1 activation. Furthermore, we demonstrate a matrix protein–dependent association of CEACAM1-L with talin, an important regulator of integrin function. Taken together, our results suggest that transmembrane CEACAM1-L expressed on endothelial cells is implicated in the activation phase of angiogenesis by affecting the cytoskeleton architecture and integrin-mediated signaling.

scholarly journals Role of the extracellular matrix protein thrombospondin in the early development of the mouse embryo.

1990 ◽  
Vol 111 (6) ◽  
pp. 2713-2723 ◽  
Author(s):  
K S O'Shea ◽  
L H Liu ◽  
L H Kinnunen ◽  
V M Dixit
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Extracellular Matrix Protein ◽  
Heparin Binding ◽  
Cell Stage ◽  
Binding Domains ◽  
Heparin Binding Domain

The distribution of the extracellular matrix protein thrombospondin (TSP) in cleavage to egg cylinder staged mouse embryos and its role in trophoblast outgrowth from cultured blastocysts were examined. TSP was present within the cytoplasm of unfertilized eggs; in fertilized one- to four-cell embryos; by the eight-cell stage, TSP was also densely deposited at cell-cell borders. In the blastocyst, although TSP was present in all three cell types; trophectoderm, endoderm, and inner cell mass (ICM), it was enriched in the ICM and at the surface of trophectoderm cells. Hatched blastocysts grown on matrix-coated coverslips formed extensive trophoblast outgrowths on TSP, grew slightly less avidly on laminin, or on a 140-kD fragment of TSP containing its COOH terminus and putative cell binding domains. There was little outgrowth on the NH2 terminus heparin-binding domain. Addition of anti-TSP antibodies (but not GRGDS) to blastocysts growing on TSP strikingly inhibited outgrowth. Consistent with its early appearance and presence in trophoblast cells during implantation, TSP may play an important role in the early events involved in mammalian embryogenesis.

scholarly journals Production of cartilage link protein by human granulosa-lutein cells

2002 ◽  
Vol 175 (2) ◽  
pp. 505-515 ◽  
Author(s):  
GW Sun ◽  
H Kobayashi ◽  
M Suzuki ◽  
N Kanayama ◽  
T Terao
Keyword(s):  
Time Course ◽  
Matrix Protein ◽  
Cultured Cells ◽  
Extracellular Matrix Protein ◽  
Regulatory Function ◽  
Human Ovary ◽  
Vitro Fertilization ◽  
Link Protein ◽  
Cell Extracts

Link protein (LP), an extracellular matrix protein in cartilage, stabilizes aggregates of hyaluronic acid (HA) and proteoglycans, including aggrecan and inter-alpha-trypsin inhibitor (ITI). We have shown previously that cartilage LP is present in the maturing rat and mouse ovary. In the present study, we have employed immunohistochemistry to examine the anatomical distribution of cartilage LP in the human ovary. The expression of cartilage LP was selectively detected in the cells within the granulosa compartment of the preovulatory dominant follicle. The HA-positive granulosa-lutein cells were found to be a cartilage LP-positive subpopulation. We subsequently studied the in vitro expression of cartilage LP in cultured human granulosa-lutein cells obtained at oocyte retrieval for in vitro fertilization. Analysis of cultured cells by enzyme-linked immunoaffinity assay, Western blotting and immunofluorescence microscopy revealed that gonadotropin stimulates cartilage LP production. Time-course studies indicated that the cartilage LP production was induced as early as with gonadotropin stimulation for 2 h, and the effect was sustained up to 8 h. Western blot analysis further revealed the presence of the macroaggregates composed of HA, ITI and cartilage LP in the gonadotropin-stimulated granulosa-lutein cell extracts. Collectively, the present results raise the possibility that cartilage LP forms extracellular structures that may have a regulatory function in the developing follicle in the human ovary.

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