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Jeffrey H Miner - One of the best experts on this subject based on the ideXlab platform.
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Laminins promote postsynaptic maturation by an autocrine mechanism at the neuromuscular junction
Journal of Cell Biology, 2008Co-Authors: Hiroshi Nishimune, Jeffrey H Miner, Gregorio Valdez, George Jarad, Casey L Moulson, Ulrich Muller, Joshua R. SanesAbstract:A prominent feature of synaptic maturation at the neuromuscular junction (NMJ) is the topological transformation of the acetylcholine receptor (AChR)-rich postsynaptic membrane from an ovoid plaque into a complex array of branches. We show here that Laminins play an autocrine role in promoting this transformation. Laminins containing the α4, α5, and β2 subunits are synthesized by muscle fibers and concentrated in the small portion of the basal lamina that passes through the synaptic cleft at the NMJ. Topological maturation of AChR clusters was delayed in targeted mutant mice lacking Laminin α5 and arrested in mutants lacking both α4 and α5. Analysis of chimeric Laminins in vivo and of mutant myotubes cultured aneurally demonstrated that the Laminins act directly on muscle cells to promote postsynaptic maturation. Immunohistochemical studies in vivo and in vitro along with analysis of targeted mutants provide evidence that Laminin-dependent aggregation of dystroglycan in the postsynaptic membrane is a key step in synaptic maturation. Another synaptically concentrated Laminin receptor, Bcam, is dispensable. Together with previous studies implicating Laminins as organizers of presynaptic differentiation, these results show that Laminins coordinate post- with presynaptic maturation.
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Laminin α5 influences the architecture of the mouse small intestine mucosa
Journal of Cell Science, 2008Co-Authors: Zhen X Mahoney, Thaddeus S Stappenbeck, Jeffrey H MinerAbstract:The mammalian intestine displays two distinct patterns of mucosal organization. The small intestine contains mucosal epithelial invaginations (the crypts of Lieberkuhn) that are continuous with evaginations (villi) into the lumen. The colon also contains crypts of Lieberkuhn, but its epithelial surface is lined by flat surface cuffs. The epithelial cells of both organs communicate with the underlying mesenchyme through a basement membrane that is composed of a variety of extracellular matrix proteins, including members of the Laminin family. The basement membranes of the small intestine and colon contain distinct Laminin subtypes; notably, the villus basement membrane is rich in Laminin α5. Here, we show that the diminution of Laminin α5 in a mouse model led to a compensatory deposition of colonic Laminins, which resulted in a transformation from a small intestinal to a colonic mucosal architecture. The alteration in mucosal architecture was associated with reduced levels of nuclear p27Kip1 – a cell-cycle regulator – and altered intestinal epithelial cell proliferation, migration and differentiation. Our results suggest that Laminin α5 has a crucial role in establishing and maintaining the specific mucosal pattern of the mouse small intestine.
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Laminin alpha 5 influences the architecture of the mouse small intestine mucosa.
Journal of cell science, 2008Co-Authors: Zhen X Mahoney, Thaddeus S Stappenbeck, Jeffrey H MinerAbstract:The mammalian intestine displays two distinct patterns of mucosal organization. The small intestine contains mucosal epithelial invaginations (the crypts of Lieberkühn) that are continuous with evaginations (villi) into the lumen. The colon also contains crypts of Lieberkühn, but its epithelial surface is lined by flat surface cuffs. The epithelial cells of both organs communicate with the underlying mesenchyme through a basement membrane that is composed of a variety of extracellular matrix proteins, including members of the Laminin family. The basement membranes of the small intestine and colon contain distinct Laminin subtypes; notably, the villus basement membrane is rich in Laminin alpha 5. Here, we show that the diminution of Laminin alpha 5 in a mouse model led to a compensatory deposition of colonic Laminins, which resulted in a transformation from a small intestinal to a colonic mucosal architecture. The alteration in mucosal architecture was associated with reduced levels of nuclear p27Kip1 - a cell-cycle regulator - and altered intestinal epithelial cell proliferation, migration and differentiation. Our results suggest that Laminin alpha 5 has a crucial role in establishing and maintaining the specific mucosal pattern of the mouse small intestine.
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Laminins and their roles in mammals
Microscopy Research and Technique, 2008Co-Authors: Jeffrey H MinerAbstract:Laminins are α-β-γ heterotrimeric components of all basement membranes. Laminins are now known to play the central role in organizing and establishing the basement membrane. The diversity of Laminins allows them to impart special structural and signaling properties to the basement membrane. Of the 12 known Laminin chain genes, 10 have been either found to be mutated in humans or experimentally mutated in mice. This has led to great progress over the last several years towards understanding both the functions of Laminins and the reasons for their great diversity. In this review, I will summarize the in vivo studies in mice and humans that have contributed to this new knowledge. Microsc. Res. Tech., 2008. © 2008 Wiley-Liss, Inc.
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Laminin functions in tissue morphogenesis
Annual Review of Cell and Developmental Biology, 2004Co-Authors: Jeffrey H Miner, Peter D YurchencoAbstract:▪ Abstract Significant advances have been made in the application of genetics to probe the functions of basement membrane Laminins. These studies have shown that different Laminin subunits profoundly affect tissue morphogenesis, starting around the time of embryonic implantation and extending through organogenesis and into the postnatal period. Collectively they have revealed common functions that include the induction and maintenance of cell polarity, the establishment of barriers between tissue compartments, the organization of cells into tissues, and the protection of adherent cells from detachment-induced cell death, anoikis. Interpreted in light of what is known about Laminin structure and self-assembly and binding activities, these advances have begun to provide insights into mechanisms of action. In this review we focus on the contributions of the Laminins in invertebrate and vertebrate tissue morphogenesis.
Mf Champliaud - One of the best experts on this subject based on the ideXlab platform.
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Posttranslational Modifications and β/γ Chain Associations of Human Laminin α1 and Laminin α5 Chains: Purification of Laminin-3 from Placenta
Experimental Cell Research, 2000Co-Authors: Mf Champliaud, Ismo Virtanen, Robert E. Burgeson, Car-fredrik Tiger, Matti Korhonen, Donald GullbergAbstract:Laminins assemble into trimers composed of alpha, beta, and gamma chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the Laminin isoforms formed by Laminin alpha1 and alpha5 chains. Comparative pulse-chase experiments and deglycosylation studies in JAR cells established that the M(r) 360,000 Laminin alpha1 chain is glycosylated into a mature M(r) 400,000 band while the M(r) 370,000 Laminin alpha5 chain is glycosylated into a M(r) 390,000 form that upon secretion is further processed into a M(r) 380,000 form. Hence, despite the shorter peptide length of alpha1 chain in comparison with the alpha5 chain, secreted alpha1 assumes a larger size in SDS-PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR Laminins identified Laminin alpha1 and Laminin alpha5 chains in Laminin-1 and Laminin-10. In placenta Laminin alpha1 chain (M(r) 400,000) and Laminin alpha5 chain (M(r) 380, 000/370,000 doublet) were found in Laminin-1/-3 and Laminin-10/-11. Immunohistochemically we could establish that the Laminin alpha1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain Laminin beta2 chains. Surprisingly, a fraction of the Laminin alpha1 chain from JAR cells and placenta could not be precipitated by antibodies to Laminin beta1-beta3 chains, possibly pointing to an unexpected complexity in the chain composition of alpha1-containing Laminin isoforms.
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Laminin Expression in Adult and Developing Retinae: Evidence of Two Novel CNS Laminins
The Journal of Neuroscience, 2000Co-Authors: Richard T. Libby, Manuel Koch, Robert E. Burgeson, Mf Champliaud, Dale D. Hunter, Thomas Claudepierre, Erin P. Gibbons, William J BrunkenAbstract:Components of the extracellular matrix exert myriad effects on tissues throughout the body. In particular, the Laminins, a family of heterotrimeric extracellular glycoproteins, have been shown to affect tissue development and integrity in such diverse organs as the kidney, lung, skin, and nervous system. Of these, we have focused on the roles that Laminins play in the differentiation and maintenance of the nervous system. Here, we examine the expression of all known Laminin chains within one component of the CNS, the retina. We find seven Laminin chains—α3, α4, α5, β2, β3, γ2, and γ3—outside the retinal basement membranes. Anatomically, these chains are coexpressed in one or both of two locations: the matrix surrounding photoreceptors and the first synaptic layer where photoreceptors synapse with retinal interneurons. Biochemically, four of these chains are coisolated from retinal extracts in two independent complexes, confirming that two novel heterotrimers—α4β2γ3 and α5β2γ3—are present in the retinal matrix. During development, all four of these chains, along with components of Laminin 5 (the α3, β3, and γ2 chains) are also expressed at sites at which they could exert important effects on photoreceptor development. Together, these data suggest the existence of two novel Laminin heterotrimers in the CNS, which we term here Laminin 14 (composed of the α4, β2, and γ3 chains) and Laminin 15 (composed of the α5, β2, and γ3 chains), and lead us to hypothesize that these Laminins, along with Laminin 5, may play roles in photoreceptor production, stability, and synaptic organization.
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characterization and expression of the Laminin γ3 chain a novel non basement membrane associated Laminin chain
Journal of Cell Biology, 1999Co-Authors: Manuel Koch, William J Brunken, Robert E. Burgeson, Dale D. Hunter, Pamela F Olson, Anne Albus, William Jin, Mf ChampliaudAbstract:Laminins are heterotrimeric molecules composed of an α, a β, and a γ chain; they have broad functional roles in development and in stabilizing epithelial structures. Here, we identified a novel Laminin, composed of known α and β chains but containing a novel γ chain, γ3. We have cloned gene encoding this chain, LAMC3, which maps to chromosome 9 at q31-34. Protein and cDNA analyses demonstrate that γ3 contains all the expected domains of a γ chain, including two consensus glycosylation sites and a putative nidogen-binding site. This suggests that γ3-containing Laminins are likely to exist in a stable matrix. Studies of the tissue distribution of γ3 chain show that it is broadly expressed in: skin, heart, lung, and the reproductive tracts. In skin, γ3 protein is seen within the basement membrane of the dermal-epidermal junction at points of nerve penetration. The γ3 chain is also a prominent element of the apical surface of ciliated epithelial cells of: lung, oviduct, epididymis, ductus deferens, and seminiferous tubules. The distribution of γ3-containing Laminins on the apical surfaces of a variety of epithelial tissues is novel and suggests that they are not found within ultrastructurally defined basement membranes. It seems likely that these apical Laminins are important in the morphogenesis and structural stability of the ciliated processes of these cells.
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human amnion contains a novel Laminin variant Laminin 7 which like Laminin 6 covalently associates with Laminin 5 to promote stable epithelial stromal attachment
Journal of Cell Biology, 1996Co-Authors: Mf Champliaud, Douglas R Keene, Patricia Rousselle, Gp Lunstrum, T. Nishiyama, Robert E. BurgesonAbstract:Stable attachment of external epithelia to the basement membrane and underlying stroma is mediated by transmembrane proteins such as the integrin alpha6beta4 and bullous pemphigoid antigen 2 within the hemidesmosomes along the basolateral surface of the epithelial cell and their ligands that include a specialized subfamily of Laminins. The Laminin 5 molecule (previously termed kalinin/nicein/epiligrin) is a member of this epithelial-specific subfamily. Laminin 5 chains are not only considerably truncated within domains III-VI, but are also extensively proteolytically processed in vitro and in vivo. As a result, the domains expected to be required for the association of Laminins with other basement membrane components are lacking in the mature Laminin 5 molecule. Therefore, the tight binding of Laminin 5 to the basement membrane may occur by a unique mechanism. To examine Laminin 5 in tissue, we chose human amnion as the source, because of its availability and the similarity of the amniotic epithelial basement membrane with that of skin. We isolated the Laminin 5 contained within the basement membrane of human amnion. In addition to monomeric Laminin 5, we find that much of the Laminin 5 isolated is covalently adducted with Laminin 6 (alpha3beta1gamma1) and a novel Laminin isotype we have termed Laminin 7 (alpha3beta2gamma1). We propose that the association between Laminin 5 and Laminins 6 and 7 is a mechanism used in amnion to allow stable association of Laminin 5 with the basement membrane. The beta2 chain is seen at the human amniotic epithelial-stromal interface and at the dermal-epidermal junction of fetal and adult bovine skin by immunofluorescence, but is not present, or only weakly present, in neonatal human skin.
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Human amnion contains a novel Laminin variant, Laminin 7, which like Laminin 6, covalently associates with Laminin 5 to promote stable epithelial-stromal attachment.
Journal of Cell Biology, 1996Co-Authors: Mf Champliaud, P. Rousselle, Gp Lunstrum, T. Nishiyama, Dr Keene, Re BurgesonAbstract:Stable attachment of external epithelia to the basement membrane and underlying stroma is mediated by transmembrane proteins such as the integrin alpha6beta4 and bullous pemphigoid antigen 2 within the hemidesmosomes along the basolateral surface of the epithelial cell and their ligands that include a specialized subfamily of Laminins. The Laminin 5 molecule (previously termed kalinin/nicein/epiligrin) is a member of this epithelial-specific subfamily. Laminin 5 chains are not only considerably truncated within domains III-VI, but are also extensively proteolytically processed in vitro and in vivo. As a result, the domains expected to be required for the association of Laminins with other basement membrane components are lacking in the mature Laminin 5 molecule. Therefore, the tight binding of Laminin 5 to the basement membrane may occur by a unique mechanism. To examine Laminin 5 in tissue, we chose human amnion as the source, because of its availability and the similarity of the amniotic epithelial basement membrane with that of skin. We isolated the Laminin 5 contained within the basement membrane of human amnion. In addition to monomeric Laminin 5, we find that much of the Laminin 5 isolated is covalently adducted with Laminin 6 (alpha3beta1gamma1) and a novel Laminin isotype we have termed Laminin 7 (alpha3beta2gamma1). We propose that the association between Laminin 5 and Laminins 6 and 7 is a mechanism used in amnion to allow stable association of Laminin 5 with the basement membrane. The beta2 chain is seen at the human amniotic epithelial-stromal interface and at the dermal-epidermal junction of fetal and adult bovine skin by immunofluorescence, but is not present, or only weakly present, in neonatal human skin.Stable attachment of external epithelia to the basement membrane and underlying stroma is mediated by transmembrane proteins such as the integrin alpha6beta4 and bullous pemphigoid antigen 2 within the hemidesmosomes along the basolateral surface of the epithelial cell and their ligands that include a specialized subfamily of Laminins. The Laminin 5 molecule (previously termed kalinin/nicein/epiligrin) is a member of this epithelial-specific subfamily. Laminin 5 chains are not only considerably truncated within domains III-VI, but are also extensively proteolytically processed in vitro and in vivo. As a result, the domains expected to be required for the association of Laminins with other basement membrane components are lacking in the mature Laminin 5 molecule. Therefore, the tight binding of Laminin 5 to the basement membrane may occur by a unique mechanism. To examine Laminin 5 in tissue, we chose human amnion as the source, because of its availability and the similarity of the amniotic epithelial basement membrane with that of skin. We isolated the Laminin 5 contained within the basement membrane of human amnion. In addition to monomeric Laminin 5, we find that much of the Laminin 5 isolated is covalently adducted with Laminin 6 (alpha3beta1gamma1) and a novel Laminin isotype we have termed Laminin 7 (alpha3beta2gamma1). We propose that the association between Laminin 5 and Laminins 6 and 7 is a mechanism used in amnion to allow stable association of Laminin 5 with the basement membrane. The beta2 chain is seen at the human amniotic epithelial-stromal interface and at the dermal-epidermal junction of fetal and adult bovine skin by immunofluorescence, but is not present, or only weakly present, in neonatal human skin.
Re Burgeson - One of the best experts on this subject based on the ideXlab platform.
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A specific and sensitive ELISA for Laminin 5.
Journal of immunological methods, 1999Co-Authors: Satoshi Amano, T Nishiyama, Re BurgesonAbstract:A sandwich ELISA for Laminin 5 was developed by using two monoclonal antibodies specific for human Laminin 5, basement membrane (BM)165 and 6F12, which were raised against the Laminin alpha3 chain and the Laminin beta3 chain, respectively. Laminin 5 was purified from squamous carcinoma cell (SCC) 25-conditioned medium, using 6F12-conjugated Sepharose. This preparation was used as the standard for the ELISA. This sandwich ELISA was sensitive enough to detect reproducibly as little as 125 pg of Laminin 5. This assay could detect Laminin 5 produced by human keratinocytes but not other Laminins produced by human fibroblasts. A portion of the Laminin 5 that was secreted by the keratinocytes was deposited around the cells, and the rest was released into the medium. To measure total synthesized Laminin 5, the intracellular and deposited Laminin 5 was solubilized by treatment with a mixture of several kinds of detergents that did not interfere with the sandwich ELISA, and the Laminin in both the culture medium and the solubilized cell layer was quantified. The amount of Laminin 5 synthesis by keratinocytes depended on the cell number, the duration of culture, and the extracellular matrix proteins on which keratinocytes were plated.
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Laminin 5 binds the NC-1 domain of type VII collagen.
Journal of Cell Biology, 1997Co-Authors: P. Rousselle, Dr Keene, Florence Ruggiero, Mf Champliaud, M. Rest, Re BurgesonAbstract:Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this structure is the major element providing for resistance to external friction. Ultrastructurally, Laminin 5 (alpha3beta3gamma2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin alpha6beta4. However, the interaction of Laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. In this study we demonstrate that monomeric Laminin 5 binds the NH2-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both Laminin 5 and type VII collagen NC-1. Laminin 6 (alpha3beta1gamma1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the beta3 and/or gamma2 chains of Laminin 5. Approximately half of the Laminin 5 solubilized from human amnion or skin is covalently complexed with Laminins 6 or 7 (alpha3beta2gamma1). The adduction occurs between the NH2 terminus of Laminin 5 and the branch point of the short arms of Laminins 6 or 7. The results are consistent with the presumed orientation of Laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa. The results also support a model predicting that monomeric Laminin 5 constitutes the anchoring filaments and bridges integrin alpha6beta4 with type VII collagen, and the Laminin 5-6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin alpha3beta1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance.Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this structure is the major element providing for resistance to external friction. Ultrastructurally, Laminin 5 (alpha3beta3gamma2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin alpha6beta4. However, the interaction of Laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. In this study we demonstrate that monomeric Laminin 5 binds the NH2-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both Laminin 5 and type VII collagen NC-1. Laminin 6 (alpha3beta1gamma1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the beta3 and/or gamma2 chains of Laminin 5. Approximately half of the Laminin 5 solubilized from human amnion or skin is covalently complexed with Laminins 6 or 7 (alpha3beta2gamma1). The adduction occurs between the NH2 terminus of Laminin 5 and the branch point of the short arms of Laminins 6 or 7. The results are consistent with the presumed orientation of Laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa. The results also support a model predicting that monomeric Laminin 5 constitutes the anchoring filaments and bridges integrin alpha6beta4 with type VII collagen, and the Laminin 5-6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin alpha3beta1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance.
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Human amnion contains a novel Laminin variant, Laminin 7, which like Laminin 6, covalently associates with Laminin 5 to promote stable epithelial-stromal attachment.
Journal of Cell Biology, 1996Co-Authors: Mf Champliaud, P. Rousselle, Gp Lunstrum, T. Nishiyama, Dr Keene, Re BurgesonAbstract:Stable attachment of external epithelia to the basement membrane and underlying stroma is mediated by transmembrane proteins such as the integrin alpha6beta4 and bullous pemphigoid antigen 2 within the hemidesmosomes along the basolateral surface of the epithelial cell and their ligands that include a specialized subfamily of Laminins. The Laminin 5 molecule (previously termed kalinin/nicein/epiligrin) is a member of this epithelial-specific subfamily. Laminin 5 chains are not only considerably truncated within domains III-VI, but are also extensively proteolytically processed in vitro and in vivo. As a result, the domains expected to be required for the association of Laminins with other basement membrane components are lacking in the mature Laminin 5 molecule. Therefore, the tight binding of Laminin 5 to the basement membrane may occur by a unique mechanism. To examine Laminin 5 in tissue, we chose human amnion as the source, because of its availability and the similarity of the amniotic epithelial basement membrane with that of skin. We isolated the Laminin 5 contained within the basement membrane of human amnion. In addition to monomeric Laminin 5, we find that much of the Laminin 5 isolated is covalently adducted with Laminin 6 (alpha3beta1gamma1) and a novel Laminin isotype we have termed Laminin 7 (alpha3beta2gamma1). We propose that the association between Laminin 5 and Laminins 6 and 7 is a mechanism used in amnion to allow stable association of Laminin 5 with the basement membrane. The beta2 chain is seen at the human amniotic epithelial-stromal interface and at the dermal-epidermal junction of fetal and adult bovine skin by immunofluorescence, but is not present, or only weakly present, in neonatal human skin.Stable attachment of external epithelia to the basement membrane and underlying stroma is mediated by transmembrane proteins such as the integrin alpha6beta4 and bullous pemphigoid antigen 2 within the hemidesmosomes along the basolateral surface of the epithelial cell and their ligands that include a specialized subfamily of Laminins. The Laminin 5 molecule (previously termed kalinin/nicein/epiligrin) is a member of this epithelial-specific subfamily. Laminin 5 chains are not only considerably truncated within domains III-VI, but are also extensively proteolytically processed in vitro and in vivo. As a result, the domains expected to be required for the association of Laminins with other basement membrane components are lacking in the mature Laminin 5 molecule. Therefore, the tight binding of Laminin 5 to the basement membrane may occur by a unique mechanism. To examine Laminin 5 in tissue, we chose human amnion as the source, because of its availability and the similarity of the amniotic epithelial basement membrane with that of skin. We isolated the Laminin 5 contained within the basement membrane of human amnion. In addition to monomeric Laminin 5, we find that much of the Laminin 5 isolated is covalently adducted with Laminin 6 (alpha3beta1gamma1) and a novel Laminin isotype we have termed Laminin 7 (alpha3beta2gamma1). We propose that the association between Laminin 5 and Laminins 6 and 7 is a mechanism used in amnion to allow stable association of Laminin 5 with the basement membrane. The beta2 chain is seen at the human amniotic epithelial-stromal interface and at the dermal-epidermal junction of fetal and adult bovine skin by immunofluorescence, but is not present, or only weakly present, in neonatal human skin.
Peter D Yurchenco - One of the best experts on this subject based on the ideXlab platform.
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chimeric protein repair of Laminin polymerization ameliorates muscular dystrophy phenotype
Journal of Clinical Investigation, 2017Co-Authors: Karen K. Mckee, Stephanie C Crosson, Judith R Reinhard, Sarina Meinen, Markus A Ruegg, Peter D YurchencoAbstract:Mutations in Laminin α2-subunit (Lmα2, encoded by LAMA2) are linked to approximately 30% of congenital muscular dystrophy cases. Mice with a homozygous mutation in Lama2 (dy2J mice) express a nonpolymerizing form of Laminin-211 (Lm211) and are a model for ambulatory-type Lmα2-deficient muscular dystrophy. Here, we developed transgenic dy2J mice with muscle-specific expression of αLNNd, a Laminin/nidogen chimeric protein that provides a missing polymerization domain. Muscle-specific expression of αLNNd in dy2J mice resulted in strong amelioration of the dystrophic phenotype, manifested by the prevention of fibrosis and restoration of forelimb grip strength. αLNNd also restored myofiber shape, size, and numbers to control levels in dy2J mice. Laminin immunostaining and quantitation of tissue extractions revealed increased Lm211 expression in αLNNd-transgenic dy2J mice. In cultured myotubes, we determined that αLNNd expression increased myotube surface accumulation of polymerization-deficient recombinant Laminins, with retention of collagen IV, reiterating the basement membrane (BM) changes observed in vivo. Laminin LN domain mutations linked to several of the Lmα2-deficient muscular dystrophies are predicted to compromise polymerization. The data herein support the hypothesis that engineered expression of αLNNd can overcome polymerization deficits to increase Laminin, stabilize BM structure, and substantially ameliorate muscular dystrophy.
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schwann cell myelination requires integration of Laminin activities
Journal of Cell Science, 2012Co-Authors: Karen K. Mckee, Zu Lin Chen, Sidney Strickland, Donghua Yang, Raj Patel, Junichi Takagi, Kiyotoshi Sekiguchi, Peter D YurchencoAbstract:Laminins promote early stages of peripheral nerve myelination by assembling basement membranes (BMs) on Schwann cell surfaces, leading to activation of β1 integrins and other receptors. The BM composition, structural bonds and ligands needed to mediate this process, however, are not well understood. Mice hypomorphic for Laminin γ1-subunit expression that assembled endoneurial BMs with reduced component density exhibited an axonal sorting defect with amyelination but normal Schwann cell proliferation, the latter unlike the null. To identify the basis for this, and to dissect participating Laminin interactions, LAMC1 gene-inactivated dorsal root ganglia were treated with recombinant Laminin-211 and -111 lacking different architecture-forming and receptor-binding activities, to induce myelination. Myelin-wrapping of axons by Schwann cells was found to require higher Laminin concentrations than either proliferation or axonal ensheathment. Laminins that were unable to polymerize through deletions that removed critical N-terminal (LN) domains, or that lacked cell-adhesive globular (LG) domains, caused reduced BMs and almost no myelination. Laminins engineered to bind weakly to α6β1 and/or α7β1 integrins through their LG domains, even though they could effectively assemble BMs, decreased myelination. Proliferation depended upon both integrin binding to LG domains and polymerization. Collectively these findings reveal that Laminins integrate scaffold-forming and cell-adhesion activities to assemble an endoneurial BM, with myelination and proliferation requiring additional α6β1/α7β1-Laminin LG domain interactions, and that a high BM ligand/structural density is needed for efficient myelination.
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scaffold forming and adhesive contributions of synthetic Laminin binding proteins to basement membrane assembly
Journal of Biological Chemistry, 2009Co-Authors: Karen K. Mckee, Stephanie Capizzi, Peter D YurchencoAbstract:Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although Laminins containing the α4 and α5 subunits are expressed in α2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient Laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (αLNNd) that adds a short arm terminus to Laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the Laminin coiled-coil dystroglycan and sulfatides. αLNNd was found to mediate Laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert α-short arm deletion-mutant Laminins LmΔαLN and LmΔαLN-L4b into polymerizing Laminins. This protein enabled polymerization-deficient Laminin but not an adhesion-deficient Laminin lacking LG domains (LmΔLG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled LmΔLG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing Laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the αLN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of Laminin deficiency states.
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role of Laminin terminal globular domains in basement membrane assembly
Journal of Biological Chemistry, 2007Co-Authors: Karen K. Mckee, Stephanie Capizzi, David G Harrison, Peter D YurchencoAbstract:Laminins contribute to basement membrane assembly through interactions of their N- and C-terminal globular domains. To further analyze this process, recombinant Laminin-111 heterotrimers with deletions and point mutations were generated by recombinant expression and evaluated for their ability to self-assemble, interact with nidogen-1 and type IV collagen, and form extracellular matrices on cultured Schwann cells by immunofluorescence and electron microscopy. Wild-type Laminin and Laminin without LG domains polymerized in contrast to Laminins with deleted alpha1-, beta1-, or gamma1-LN domains or with duplicated beta1- or alpha1-LN domains. Laminins with a full complement of LN and LG domains accumulated on cell surfaces substantially above those lacking either LN or LG domains and formed a lamina densa. Accumulation of type IV collagen onto the cell surface was found to require Laminin with separate contributions arising from the presence of Laminin LN domains, nidogen-1, and the nidogen-binding site in Laminin. Collectively, the data support the hypothesis that basement membrane assembly depends on Laminin self-assembly through formation of alpha-, beta-, and gamma-LN domain complexes and LG-mediated cell surface anchorage. Furthermore, type IV collagen recruitment into the Laminin extracellular matrices appears to be mediated through a nidogen bridge with a lesser contribution arising from a direct interaction with Laminin.
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Laminin functions in tissue morphogenesis
Annual Review of Cell and Developmental Biology, 2004Co-Authors: Jeffrey H Miner, Peter D YurchencoAbstract:▪ Abstract Significant advances have been made in the application of genetics to probe the functions of basement membrane Laminins. These studies have shown that different Laminin subunits profoundly affect tissue morphogenesis, starting around the time of embryonic implantation and extending through organogenesis and into the postnatal period. Collectively they have revealed common functions that include the induction and maintenance of cell polarity, the establishment of barriers between tissue compartments, the organization of cells into tissues, and the protection of adherent cells from detachment-induced cell death, anoikis. Interpreted in light of what is known about Laminin structure and self-assembly and binding activities, these advances have begun to provide insights into mechanisms of action. In this review we focus on the contributions of the Laminins in invertebrate and vertebrate tissue morphogenesis.
Robert E. Burgeson - One of the best experts on this subject based on the ideXlab platform.
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Posttranslational Modifications and β/γ Chain Associations of Human Laminin α1 and Laminin α5 Chains: Purification of Laminin-3 from Placenta
Experimental Cell Research, 2000Co-Authors: Mf Champliaud, Ismo Virtanen, Robert E. Burgeson, Car-fredrik Tiger, Matti Korhonen, Donald GullbergAbstract:Laminins assemble into trimers composed of alpha, beta, and gamma chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the Laminin isoforms formed by Laminin alpha1 and alpha5 chains. Comparative pulse-chase experiments and deglycosylation studies in JAR cells established that the M(r) 360,000 Laminin alpha1 chain is glycosylated into a mature M(r) 400,000 band while the M(r) 370,000 Laminin alpha5 chain is glycosylated into a M(r) 390,000 form that upon secretion is further processed into a M(r) 380,000 form. Hence, despite the shorter peptide length of alpha1 chain in comparison with the alpha5 chain, secreted alpha1 assumes a larger size in SDS-PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR Laminins identified Laminin alpha1 and Laminin alpha5 chains in Laminin-1 and Laminin-10. In placenta Laminin alpha1 chain (M(r) 400,000) and Laminin alpha5 chain (M(r) 380, 000/370,000 doublet) were found in Laminin-1/-3 and Laminin-10/-11. Immunohistochemically we could establish that the Laminin alpha1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain Laminin beta2 chains. Surprisingly, a fraction of the Laminin alpha1 chain from JAR cells and placenta could not be precipitated by antibodies to Laminin beta1-beta3 chains, possibly pointing to an unexpected complexity in the chain composition of alpha1-containing Laminin isoforms.
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Laminin Expression in Adult and Developing Retinae: Evidence of Two Novel CNS Laminins
The Journal of Neuroscience, 2000Co-Authors: Richard T. Libby, Manuel Koch, Robert E. Burgeson, Mf Champliaud, Dale D. Hunter, Thomas Claudepierre, Erin P. Gibbons, William J BrunkenAbstract:Components of the extracellular matrix exert myriad effects on tissues throughout the body. In particular, the Laminins, a family of heterotrimeric extracellular glycoproteins, have been shown to affect tissue development and integrity in such diverse organs as the kidney, lung, skin, and nervous system. Of these, we have focused on the roles that Laminins play in the differentiation and maintenance of the nervous system. Here, we examine the expression of all known Laminin chains within one component of the CNS, the retina. We find seven Laminin chains—α3, α4, α5, β2, β3, γ2, and γ3—outside the retinal basement membranes. Anatomically, these chains are coexpressed in one or both of two locations: the matrix surrounding photoreceptors and the first synaptic layer where photoreceptors synapse with retinal interneurons. Biochemically, four of these chains are coisolated from retinal extracts in two independent complexes, confirming that two novel heterotrimers—α4β2γ3 and α5β2γ3—are present in the retinal matrix. During development, all four of these chains, along with components of Laminin 5 (the α3, β3, and γ2 chains) are also expressed at sites at which they could exert important effects on photoreceptor development. Together, these data suggest the existence of two novel Laminin heterotrimers in the CNS, which we term here Laminin 14 (composed of the α4, β2, and γ3 chains) and Laminin 15 (composed of the α5, β2, and γ3 chains), and lead us to hypothesize that these Laminins, along with Laminin 5, may play roles in photoreceptor production, stability, and synaptic organization.
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characterization and expression of the Laminin γ3 chain a novel non basement membrane associated Laminin chain
Journal of Cell Biology, 1999Co-Authors: Manuel Koch, William J Brunken, Robert E. Burgeson, Dale D. Hunter, Pamela F Olson, Anne Albus, William Jin, Mf ChampliaudAbstract:Laminins are heterotrimeric molecules composed of an α, a β, and a γ chain; they have broad functional roles in development and in stabilizing epithelial structures. Here, we identified a novel Laminin, composed of known α and β chains but containing a novel γ chain, γ3. We have cloned gene encoding this chain, LAMC3, which maps to chromosome 9 at q31-34. Protein and cDNA analyses demonstrate that γ3 contains all the expected domains of a γ chain, including two consensus glycosylation sites and a putative nidogen-binding site. This suggests that γ3-containing Laminins are likely to exist in a stable matrix. Studies of the tissue distribution of γ3 chain show that it is broadly expressed in: skin, heart, lung, and the reproductive tracts. In skin, γ3 protein is seen within the basement membrane of the dermal-epidermal junction at points of nerve penetration. The γ3 chain is also a prominent element of the apical surface of ciliated epithelial cells of: lung, oviduct, epididymis, ductus deferens, and seminiferous tubules. The distribution of γ3-containing Laminins on the apical surfaces of a variety of epithelial tissues is novel and suggests that they are not found within ultrastructurally defined basement membranes. It seems likely that these apical Laminins are important in the morphogenesis and structural stability of the ciliated processes of these cells.
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A specific and sensitive ELISA for Laminin 5.
Journal of Immunological Methods, 1999Co-Authors: Satoshi Amano, T. Nishiyama, Robert E. BurgesonAbstract:Abstract A sandwich ELISA for Laminin 5 was developed by using two monoclonal antibodies specific for human Laminin 5, basement membrane (BM)165 and 6F12, which were raised against the Laminin α3 chain and the Laminin β3 chain, respectively. Laminin 5 was purified from squamous carcinoma cell (SCC) 25-conditioned medium, using 6F12-conjugated Sepharose. This preparation was used as the standard for the ELISA. This sandwich ELISA was sensitive enough to detect reproducibly as little as 125 pg of Laminin 5. This assay could detect Laminin 5 produced by human keratinocytes but not other Laminins produced by human fibroblasts. A portion of the Laminin 5 that was secreted by the keratinocytes was deposited around the cells, and the rest was released into the medium. To measure total synthesized Laminin 5, the intracellular and deposited Laminin 5 was solubilized by treatment with a mixture of several kinds of detergents that did not interfere with the sandwich ELISA, and the Laminin in both the culture medium and the solubilized cell layer was quantified. The amount of Laminin 5 synthesis by keratinocytes depended on the cell number, the duration of culture, and the extracellular matrix proteins on which keratinocytes were plated.
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Self-assembly of Laminin Isoforms
Journal of Biological Chemistry, 1997Co-Authors: Yunhui Cheng, Robert E. Burgeson, Peter D YurchencoAbstract:Abstract The α, β, and γ subunits of basement membrane Laminins can combine into different heterotrimeric molecules with either three full short arms (e.g. Laminins-1–4), or molecules containing one (Laminins-6–9) or more (Laminin-5) short arm truncations. Laminin-1 (α1β1γ1), self-assembles through a calcium-dependent thermal gelation requiring binding interactions between N-terminal short arm domains, forming a meshwork polymer thought to contribute to basement membrane architecture (Yurchenco, P. D., and Cheng, Y. S. (1993) J. Biol. Chem. 268, 17286–17299). However, it has been unclear whether other isoforms share this property, and if so, which ones. To begin to address this, we evaluated Laminin-2 (α2β1γ1), Laminin-4 (α2β2γ1), Laminin-5 (α3Aβ3γ2), and Laminin-6 (α3Aβ1γ1). The first two isoforms were found to self-aggregate in a concentration- and temperature-dependent manner and a close self-assembly relationship among Laminins-1, -2, and -4 were demonstrated by: (a) polymerization of all three proteins was inhibited by EDTA and Laminin-1 short arm fragments, (b) polymerization of Laminin-1 was inhibited by fragments of Laminins-2 and -4, (c) Laminin-2 and, to a lesser degree, Laminin-4, even well below their own critical concentration, co-aggregated with Laminin-1, evidence for co-polymerization. Laminin-5, on the other hand, neither polymerized nor co-polymerized with Laminin-1. Laminin-6 failed to co-aggregate with Laminin-1 at all concentrations evaluated, evidence for a lack of a related self-assembly activity. The data support the hypothesis that all three short arms are required for self-assembly and suggest that the short arm domain structure of Laminin isoforms affect their architecture-forming properties in basement membranes.
