The major laminin receptor of mouse embryonic stem cells is a novel isoform of the alpha 6 beta 1 integrin.

Laminin is the first extracellular matrix protein expressed in the developing mouse embryo. It is known to influence morphogenesis and affect cell migration and polarization. Several laminin receptors are included in the integrin family of extracellular matrix receptors. Ligand binding by integrin heterodimers results in signal transduction events controlling cell motility. We report that the major laminin receptor on murine embryonic stem (ES) cells is the integrin heterodimer alpha 6 beta 1, an important receptor for laminin in neurons, lymphocytes, macrophages, fibroblasts, platelets and other cell types. However, the cytoplasmic domain of the ES cell alpha 6 (alpha 6 B) differs totally from the reported cytoplasmic domain amino acid sequence of alpha 6 (alpha 6 A). Comparisons of alpha 6 cDNAs from ES cells and other cells suggest that the alpha 6 A and alpha 6 B cytoplasmic domains derive from alternative mRNA splicing. Anti-peptide antibodies to alpha 6 A are unreactive with ES cells, but react with mouse melanoma cells and embryonic fibroblasts. When ES cells are cultured under conditions that permit their differentiation, they become positive for alpha 6 A, concurrent with the morphologic appearance of differentiated cell types. Thus, expression of the alpha 6 B beta 1 laminin receptor may be favored in undifferentiated, totipotent cells, while the expression of alpha 6 A beta 1 receptor occurs in committed lineages. While the functions of integrin alpha chain cytoplasmic domains are not understood, it is possible that they contribute to transferring signals to the cell interior, e.g., by delivering cytoskeleton organizing signals in response to integrin engagement with extracellular matrix ligands. It is therefore reasonable to propose that the cellular responses to laminin may vary, according to what alpha subunit isoform (alpha 6 A or alpha 6 B) is expressed as part of the alpha 6 beta 1 laminin receptor. The switch from alpha 6 B to alpha 6 A, if confirmed in early embryos, could then be of striking potential relevance to the developmental role of laminin.

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Recombinant Extracellular Matrix Protein Fragments Support Human Embryonic Stem Cell Chondrogenesis

Tissue Engineering Part A ◽

10.1089/ten.tea.2017.0285 ◽

2018 ◽

Vol 24 (11-12) ◽

pp. 968-978 ◽

Cited By ~ 11

Author(s):

Aixin Cheng ◽

Stuart A. Cain ◽

Pinyuan Tian ◽

Andrew K. Baldwin ◽

Paweena Uppanan ◽

...

Keyword(s):

Extracellular Matrix ◽

Stem Cell ◽

Embryonic Stem Cell ◽

Human Embryonic Stem Cell ◽

Matrix Protein ◽

Embryonic Stem ◽

Extracellular Matrix Protein ◽

Protein Fragments

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

The Journal of Cell Biology ◽

10.1083/jcb.111.6.2713 ◽

1990 ◽

Vol 111 (6) ◽

pp. 2713-2723 ◽

Cited By ~ 39

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.

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Integrin α6Aβ1 Induces CD81-dependent Cell Motility without Engaging the Extracellular Matrix Migration Substrate

Molecular Biology of the Cell ◽

10.1091/mbc.8.11.2253 ◽

1997 ◽

Vol 8 (11) ◽

pp. 2253-2265 ◽

Cited By ~ 45

Author(s):

Susan Z. Domanico ◽

Anthony J. Pelletier ◽

Wendy L. Havran ◽

Vito Quaranta

Keyword(s):

Extracellular Matrix ◽

Cell Motility ◽

Es Cells ◽

Embryonic Stem ◽

Inhibitory Effect ◽

Laminin Receptor ◽

Cross Linking ◽

Integrin Subunit ◽

Dependent Cell ◽

Underlying Mechanisms

It is well established that integrins and extracellular matrix (ECM) play key roles in cell migration, but the underlying mechanisms are poorly defined. We describe a novel mechanism whereby the integrin α6β1, a laminin receptor, can affect cell motility and induce migration onto ECM substrates with which it is not engaged. By using DNA-mediated gene transfer, we expressed the human integrin subunit α6A in murine embryonic stem (ES) cells. ES cells expressing α6A (ES6A) at the surface dimerized with endogenous β1, extended numerous filopodia and lamellipodia, and were intensely migratory in haptotactic assays on laminin (LN)-1. Transfected α6A was responsible for these effects, because cells transfected with control vector or α6B, a cytoplasmic domain α6 isoform, displayed compact morphology and no migration, like wild-type ES cells. The ES6A migratory phenotype persisted on fibronectin (Fn) and Ln-5. Adhesion inhibition assays indicated that α6β1 did not contribute detectably to adhesion to these substrates in ES cells. However, anti-α6 antibodies completely blocked migration of ES6A cells on Fn or Ln-5. Control experiments with monensin and anti-ECM antibodies indicated that this inhibition could not be explained by deposition of an α6β1 ligand (e.g., Ln-1) by ES cells. Cross-linking with secondary antibody overcame the inhibitory effect of anti-α6 antibodies, restoring migration or filopodia extension on Fn and Ln-5. Thus, to induce migration in ES cells, α6Aβ1 did not have to engage with an ECM ligand but likely participated in molecular interactions sensitive to anti-α6β1 antibody and mimicked by cross-linking. Antibodies to the tetraspanin CD81 inhibited α6Aβ1-induced migration but had no effect on ES cell adhesion. It is known that CD81 is physically associated with α6β1, therefore our results suggest a mechanism by which interactions between α6Aβ1 and CD81 may up-regulate cell motility, affecting migration mediated by other integrins.

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Adhesomes: specific granules containing receptors for laminin, C3bi/fibrinogen, fibronectin, and vitronectin in human polymorphonuclear leukocytes and monocytes.

The Journal of Cell Biology ◽

10.1083/jcb.109.6.3169 ◽

1989 ◽

Vol 109 (6) ◽

pp. 3169-3182 ◽

Cited By ~ 99

Author(s):

I I Singer ◽

S Scott ◽

D W Kawka ◽

D M Kazazis

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Leukocyte Adhesion ◽

Specific Binding ◽

Laminin Receptor ◽

Extracellular Matrix Protein ◽

Outer Cell ◽

Fibronectin Receptor ◽

Specific Granules ◽

Protein Receptors

We have localized several major extracellular matrix protein receptors in the specific granules of human polymorphonuclear (PMN) and monocytic leukocytes using double label immunoelectron microscopy (IEM) with ultrathin frozen sections and colloidal-gold conjugates. Rabbit antibodies to 67-kD human laminin receptor (LNR) were located on the inner surface of the specific granule membrane and within its internal matrix. LNR antigens co-distributed with lactoferrin, a marker of specific granules, but did not co-localize with elastase in azurophilic granules of PMNs. Further, CD11b/CD18 (leukocyte receptor for C3bi, fibrinogen, endothelial cells, and endotoxin), mammalian fibronectin receptor (FNR), and vitronectin receptor (VNR) antigens were also co-localized with LNR in PMN specific granules. A similar type of granule was found in monocytes which stained for LNR, FNR, VNR, CD18, and lysozyme. Activation of PMNs with either PMA, f-met-leu-phe (fMLP), tumor necrosis factor (TNF), or monocytic leukocytes with lipopolysaccharide (LPS), induced fusion of specific granules with the cell membrane and expression of both LNR and CD18 antigens on the outer cell surface. Further, stimulation led to augmented PMN adhesion on LN substrata, and six- to eightfold increases in specific binding of soluble LN that was inhibited by LNR antibody. These results indicate that four types of extracellular matrix receptors are located in leukocyte specific granules, and suggest that up-regulation of these receptors during inflammation may mediate leukocyte adhesion and extravasation. We have thus termed leukocyte specific granules adhesomes.

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Pericardin, aDrosophilatype IV collagen-like protein is involved in the morphogenesis and maintenance of the heart epithelium during dorsal ectoderm closure

Development ◽

10.1242/dev.129.13.3241 ◽

2002 ◽

Vol 129 (13) ◽

pp. 3241-3253 ◽

Cited By ~ 7

Author(s):

Aymeric Chartier ◽

Stéphane Zaffran ◽

Martine Astier ◽

Michel Sémériva ◽

Danielle Gratecos

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Cell Types ◽

Tube Formation ◽

Extracellular Matrix Protein ◽

Heart Cells ◽

Dorsal Closure ◽

Heart Tube ◽

Pericardial Cells ◽

Dorsal Ectoderm

The steps that lead to the formation of a single primitive heart tube are highly conserved in vertebrate and invertebrate embryos. Concerted migration of the two lateral cardiogenic regions of the mesoderm and endoderm (or ectoderm in invertebrates) is required for their fusion at the midline of the embryo. Morphogenetic signals are involved in this process and the extracellular matrix has been proposed to serve as a link between the two layers of cells.Pericardin (Prc), a novel Drosophila extracellular matrix protein is a good candidate to participate in heart tube formation. The protein has the hallmarks of a type IV collagen α-chain and is mainly expressed in the pericardial cells at the onset of dorsal closure. As dorsal closure progresses, Pericardin expression becomes concentrated at the basal surface of the cardioblasts and around the pericardial cells, in close proximity to the dorsal ectoderm. Pericardin is absent from the lumen of the dorsal vessel.Genetic evidence suggests that Prc promotes the proper migration and alignment of heart cells. Df(3)vin6 embryos, as well as embryos in which prc has been silenced via RNAi, exhibit similar and significant defects in the formation of the heart epithelium. In these embryos, the heart epithelium appears disorganized during its migration to the dorsal midline. By the end of embryonic development, cardial and pericardial cells are misaligned such that small clusters of both cell types appear in the heart; these clusters of cells are associated with holes in the walls of the heart. A prc transgene can partially rescue each of these phenotypes, suggesting that prc regulates these events. Our results support, for the first time, the function of a collagen-like protein in the coordinated migration of dorsal ectoderm and heart cells.

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The extracellular matrix protein agrin is essential for epicardial epithelial-to-mesenchymal transition during heart development

Development ◽

10.1242/dev.197525 ◽

2021 ◽

Vol 148 (9) ◽

Author(s):

Xin Sun ◽

Sophia Malandraki-Miller ◽

Tahnee Kennedy ◽

Elad Bassat ◽

Konstantinos Klaourakis ◽

...

Keyword(s):

Extracellular Matrix ◽

Basement Membrane ◽

Heart Development ◽

Matrix Protein ◽

Wilms Tumor ◽

Embryonic Stem ◽

Focal Adhesions ◽

Extracellular Matrix Protein ◽

Mesenchymal Transition ◽

Epicardial Cells

ABSTRACT During heart development, epicardial cells residing within the outer layer undergo epithelial-mesenchymal transition (EMT) and migrate into the underlying myocardium to support organ growth and morphogenesis. Disruption of epicardial EMT results in embryonic lethality, yet its regulation is poorly understood. Here, we report epicardial EMT within the mesothelial layer of the mouse embryonic heart at ultra-high resolution using scanning electron microscopy combined with immunofluorescence analyses. We identified morphologically active EMT regions that associated with key components of the extracellular matrix, including the basement membrane-associated proteoglycan agrin. Deletion of agrin resulted in impaired EMT and compromised development of the epicardium, accompanied by downregulation of Wilms’ tumor 1. Agrin enhanced EMT in human embryonic stem cell-derived epicardial-like cells by decreasing β-catenin and promoting pFAK localization at focal adhesions, and promoted the aggregation of dystroglycan within the Golgi apparatus in murine epicardial cells. Loss of agrin resulted in dispersal of dystroglycan in vivo, disrupting basement membrane integrity and impairing EMT. Our results provide new insights into the role of the extracellular matrix in heart development and implicate agrin as a crucial regulator of epicardial EMT.

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Extracellular matrix protein-1 secretory isoform promotes ovarian cancer through increasing alternative mRNA splicing and stemness

Nature Communications ◽

10.1038/s41467-021-24315-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Huijing Yin ◽

Jingshu Wang ◽

Hui Li ◽

Yinjue Yu ◽

Xiaoling Wang ◽

...

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Mrna Splicing ◽

Extracellular Matrix Protein ◽

Good Survival ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Multiple Organs ◽

Extracellular Matrix Protein 1 ◽

Alternative Mrna Splicing ◽

Nuclear Ribonucleoprotein

AbstractExtracellular matrix protein-1 (ECM1) promotes tumorigenesis in multiple organs but the mechanisms associated to ECM1 isoform subtypes have yet to be clarified. We report in this study that the secretory ECM1a isoform induces tumorigenesis through the GPR motif binding to integrin αXβ2 and the activation of AKT/FAK/Rho/cytoskeleton signaling. The ATP binding cassette subfamily G member 1 (ABCG1) transduces the ECM1a-integrin αXβ2 interactive signaling to facilitate the phosphorylation of AKT/FAK/Rho/cytoskeletal molecules and to confer cancer cell cisplatin resistance through up-regulation of the CD326-mediated cell stemness. On the contrary, the non-secretory ECM1b isoform binds myosin and blocks its phosphorylation, impairing cytoskeleton-mediated signaling and tumorigenesis. Moreover, ECM1a induces the expression of the heterogeneous nuclear ribonucleoprotein L like (hnRNPLL) protein to favor the alternative mRNA splicing generating ECM1a. ECM1a, αXβ2, ABCG1 and hnRNPLL higher expression associates with poor survival, while ECM1b higher expression associates with good survival. These results highlight ECM1a, integrin αXβ2, hnRNPLL and ABCG1 as potential targets for treating cancers associated with ECM1-activated signaling.

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1280: Increased Susceptibility to Genitovaginal Prolapse Associated with a Polymorphism in the Promoter of the Extracellular Matrix Protein LAMC1

The Journal of Urology ◽

10.1016/s0022-5347(18)31494-0 ◽

2007 ◽

Vol 177 (4S) ◽

pp. 421-422

Author(s):

Ganka Nikolova ◽

Christian O. Twiss ◽

Hane Lee ◽

Nelson Stanley ◽

Janet Sinsheimer ◽

...

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Extracellular Matrix Protein ◽

Increased Susceptibility

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Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11438-0 ◽

2021 ◽

Author(s):

Aniel Moya-Torres ◽

Monika Gupta ◽

Fabian Heide ◽

Natalie Krahn ◽

Scott Legare ◽

...

Keyword(s):

Extracellular Matrix ◽

Hollow Fiber ◽

Mammalian Cells ◽

Matrix Protein ◽

Continuous Production ◽

Extracellular Matrix Protein ◽

Close Monitoring ◽

High Quality ◽

Biolayer Interferometry ◽

Hollow Fiber Bioreactor

Abstract The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. Key points • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality Graphical abstract

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