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Cay M. Kielty - One of the best experts on this subject based on the ideXlab platform.
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2020Co-Authors: Teresa Massam-wu, Amanda Mcgovern, Maybo Chiu, Rawshan Choudhury, Andrew K Baldwin, Shazia S Chaudhry, Clair Baldock, Adrian C Shuttleworth, Cay M. KieltyAbstract:Summary Control of the bioavailability of the growth factor TGF is essential for tissue formation and homeostasis, yet precisely how latent TGF is incorporated into the extracellular matrix is unknown. Here, we show that deposition of a large latent TGF complex (LLC), which contains latent TGF-binding protein 1 (LTBP-1), is directly dependent on the pericellular assembly of fibrillin Microfibrils, which interact with fibronectin during higher-order fibrillogenesis. LTBP-1 formed pericellular arrays that colocalized with Microfibrils, whereas fibrillin knockdown inhibited fibrillar LTBP-1 and/or LLC deposition. Blocking 51 integrin or supplementing cultures with heparin, which both inhibited Microfibril assembly, disrupted LTBP-1 deposition and enhanced Smad2 phosphorylation. Full-length LTBP-1 bound only weakly to N-terminal pro-fibrillin-1, but this association was strongly enhanced by heparin. The Microfibrilassociated glycoprotein MAGP-1 (MFAP-2) inhibited LTBP-1 binding to fibrillin-1 and stimulated Smad2 phosphorylation. By contrast, fibulin-4, which interacted strongly with full-length LTBP-1, did not induce Smad2 phosphorylation. Thus, LTBP-1 and/or LLC deposition is dependent on pericellular Microfibril assembly and is governed by complex interactions between LTBP-1, heparan sulfate, fibrillin-1 and Microfibril-associated molecules. In this way, Microfibrils control TGF bioavailability
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epithelial mesenchymal status influences how cells deposit fibrillin Microfibrils
2014Co-Authors: Andrew K Baldwin, Alan R F Godwin, Rachel Lennon, Stuart A Cain, Catherine L R Merry, Cay M. KieltyAbstract:Here, we show that epithelial–mesenchymal status influences how cells deposit extracellular matrix. Retinal pigmented epithelial (RPE) cells that expressed high levels of E-cadherin and had cell–cell junctions rich in zona occludens (ZO)-1, β-catenin and heparan sulfate, required syndecan-4 but not fibronectin or protein kinase C α (PKCα) to assemble extracellular matrix (fibrillin Microfibrils and perlecan). In contrast, RPE cells that strongly expressed mesenchymal smooth muscle α-actin but little ZO-1 or E-cadherin, required fibronectin (like fibroblasts) and PKCα, but not syndecan-4. Integrins α5β1 and/or α8β1 and actomyosin tension were common requirements for Microfibril deposition, as was heparan sulfate biosynthesis. TGFβ, which stimulates epithelial–mesenchymal transition, altered gene expression and overcame the dependency on syndecan-4 for Microfibril deposition in epithelial RPE cells, whereas blocking cadherin interactions disrupted Microfibril deposition. Renal podocytes had a transitional phenotype with pericellular β-catenin but little ZO-1; they required syndecan-4 and fibronectin for efficient Microfibril deposition. Thus, epithelial–mesenchymal status modulates Microfibril deposition.
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assembly of fibrillin Microfibrils governs extracellular deposition of latent tgfβ
2010Co-Authors: Teresa Massamwu, Amanda Mcgovern, Maybo Chiu, Rawshan Choudhury, Andrew K Baldwin, Shazia S Chaudhry, Clair Baldock, Cay M. KieltyAbstract:Control of the bioavailability of the growth factor TGFβ is essential for tissue formation and homeostasis, yet precisely how latent TGFβ is incorporated into the extracellular matrix is unknown. Here, we show that deposition of a large latent TGFβ complex (LLC), which contains latent TGFβ-binding protein 1 (LTBP-1), is directly dependent on the pericellular assembly of fibrillin Microfibrils, which interact with fibronectin during higher-order fibrillogenesis. LTBP-1 formed pericellular arrays that colocalized with Microfibrils, whereas fibrillin knockdown inhibited fibrillar LTBP-1 and/or LLC deposition. Blocking α5β1 integrin or supplementing cultures with heparin, which both inhibited Microfibril assembly, disrupted LTBP-1 deposition and enhanced Smad2 phosphorylation. Full-length LTBP-1 bound only weakly to N-terminal pro-fibrillin-1, but this association was strongly enhanced by heparin. The Microfibril-associated glycoprotein MAGP-1 (MFAP-2) inhibited LTBP-1 binding to fibrillin-1 and stimulated Smad2 phosphorylation. By contrast, fibulin-4, which interacted strongly with full-length LTBP-1, did not induce Smad2 phosphorylation. Thus, LTBP-1 and/or LLC deposition is dependent on pericellular Microfibril assembly and is governed by complex interactions between LTBP-1, heparan sulfate, fibrillin-1 and Microfibril-associated molecules. In this way, Microfibrils control TGFβ bioavailability.
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Fibrillin-1 Microfibril deposition is dependent on fibronectin assembly.
2008Co-Authors: Rachel Kinsey, Matthew R Williamson, Shazia Chaudhry, Amanda Mcgovern, Seiichiro Takahashi, Kieran T Mellody, C Adrian Shuttleworth, Cay M. KieltyAbstract:Newly deposited Microfibrils strongly colocalise with fibronectin in primary fibroblasts. Microfibril formation is grossly inhibited by fibronectin depletion, but rescued by supplementation with exogenous cellular fibronectin. As integrin receptors are key determinants of fibronectin assembly, we investigated whether they also influenced Microfibril deposition. Analysis of beta1-integrin-receptor-null fibroblasts, blockage of cell surface integrin receptors that regulate fibronectin assembly and disruption of Rho kinase all result in suppressed deposition of both fibronectin and Microfibrils. Antibody activation of beta1 integrins in fibronectin-depleted cultures is insufficient to rescue Microfibril assembly. In fibronectin(RGE/RGE) mutant mouse fibroblast cultures, which do not engage alpha5beta1 integrin, extracellular assembly of both fibronectin and Microfibrils is markedly reduced. Thus, pericellular Microfibril assembly is regulated by fibronectin fibrillogenesis.
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Fibrillin-1 Microfibril deposition is dependent on fibronectin assembly
2008Co-Authors: Rachel Kinsey, Matthew R Williamson, Amanda Mcgovern, Seiichiro Takahashi, Kieran T Mellody, Shazia S Chaudhry, C Adrian Shuttleworth, Cay M. KieltyAbstract:Newly deposited Microfibrils strongly colocalise with fibronectin in primary fibroblasts. Microfibril formation is grossly inhibited by fibronectin depletion, but rescued by supplementation with exogenous cellular fibronectin. As integrin receptors are key determinants of fibronectin assembly, we investigated whether they also influenced Microfibril deposition. Analysis of β1-integrin-receptor-null fibroblasts, blockage of cell surface integrin receptors that regulate fibronectin assembly and disruption of Rho kinase all result in suppressed deposition of both fibronectin and Microfibrils. Antibody activation of β1 integrins in fibronectin-depleted cultures is insufficient to rescue Microfibril assembly. In fibronectin RGE/RGE mutant mouse fibroblast cultures, which do not engage α5β1 integrin, extracellular assembly of both fibronectin and Microfibrils is markedly reduced. Thus, pericellular Microfibril assembly is regulated by fibronectin fibrillogenesis.
Penny A Handford - One of the best experts on this subject based on the ideXlab platform.
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assembly assay identifies a critical region of human fibrillin 1 required for 10 12 nm diameter Microfibril biogenesis
2021Co-Authors: Sacha A. Jensen, Ondine Atwa, Penny A HandfordAbstract:The human FBN1 gene encodes fibrillin-1 (FBN1); the main component of the 10 - 12 nm diameter extracellular matrix Microfibrils. Marfan syndrome (MFS) is a common inherited connective tissue disorder, caused by FBN1 mutations. It features a wide spectrum of disease severity, from mild cases to the lethal neonatal form (nMFS), that is yet to be explained at the molecular level. Mutations associated with nMFS generally affect a region of FBN1 between domains TB3-cbEGF18 — the "neonatal region". To gain insight into the process of fibril assembly and increase our understanding of the mechanisms determining disease severity in MFS, we compared the secretion and assembly properties of FBN1 variants containing nMFS-associated substitutions with variants associated with milder, classical MFS (cMFS). In the majority of cases, both nMFS- and cMFS-associated neonatal region variants were secreted at levels comparable to wild type. Microfibril incorporation by the nMFS variants was greatly reduced or absent compared to the cMFS forms, however, suggesting that nMFS substitutions disrupt a previously undefined site of Microfibril assembly. Additional analysis of a domain deletion variant caused by exon skipping also indicates that register in the neonatal region is likely to be critical for assembly. These data demonstrate for the first time new requirements for Microfibril biogenesis and identify at least two distinct molecular mechanisms associated with disease substitutions in the TB3-cbEGF18 region; incorporation of mutant FBN1 into Microfibrils changing their integral properties (cMFS) or the blocking of wild type FBN1 assembly by mutant molecules that prevents late-stage lateral assembly (nMFS).
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new insights into the structure assembly and biological roles of 10 12 nm connective tissue Microfibrils from fibrillin 1 studies
2016Co-Authors: Sacha A. Jensen, Penny A HandfordAbstract:The 10-12 nm diameter Microfibrils of the extracellular matrix (ECM) impart both structural and regulatory properties to load-bearing connective tissues. The main protein component is the calcium-dependent glycoprotein fibrillin, which assembles into Microfibrils at the cell surface in a highly regulated process involving specific proteolysis, multimerization and glycosaminoglycan interactions. In higher metazoans, Microfibrils act as a framework for elastin deposition and modification, resulting in the formation of elastic fibres, but they can also occur in elastin-free tissues where they perform structural roles. Fibrillin Microfibrils are further engaged in a number of cell matrix interactions such as with integrins, bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-β (TGFβ). Fibrillin-1 (FBN1) mutations are associated with a range of heritable connective disorders, including Marfan syndrome (MFS) and the acromelic dysplasias, suggesting that the roles of 10-12 nm diameter Microfibrils are pleiotropic. In recent years the use of molecular, cellular and whole-organism studies has revealed that the Microfibril is not just a structural component of the ECM, but through its network of cell and matrix interactions it can exert profound regulatory effects on cell function. In this review we assess what is known about the molecular properties of fibrillin that enable it to assemble into the 10-12 nm diameter Microfibril and perform such diverse roles.
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new insights into the structure assembly and biological roles of 10 12 nm connective tissue Microfibrils from fibrillin 1 studies
2016Co-Authors: Sacha A. Jensen, Penny A HandfordAbstract:The 10–12 nm diameter Microfibrils of the extracellular matrix (ECM) impart both structural and regulatory properties to load-bearing connective tissues. The main protein component is the calcium-dependent glycoprotein fibrillin, which assembles into Microfibrils at the cell surface in a highly regulated process involving specific proteolysis, multimerization and glycosaminoglycan interactions. In higher metazoans, Microfibrils act as a framework for elastin deposition and modification, resulting in the formation of elastic fibres, but they can also occur in elastin-free tissues where they perform structural roles. Fibrillin Microfibrils are further engaged in a number of cell matrix interactions such as with integrins, bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-β (TGFβ). Fibrillin-1 ( FBN1 ) mutations are associated with a range of heritable connective disorders, including Marfan syndrome (MFS) and the acromelic dysplasias, suggesting that the roles of 10–12 nm diameter Microfibrils are pleiotropic. In recent years the use of molecular, cellular and whole-organism studies has revealed that the Microfibril is not just a structural component of the ECM, but through its network of cell and matrix interactions it can exert profound regulatory effects on cell function. In this review we assess what is known about the molecular properties of fibrillin that enable it to assemble into the 10–12 nm diameter Microfibril and perform such diverse roles. * ADAMTS(L), : a disintegrin and metalloproteinase with thrombospondin motifs(-like); BMP, : bone morphogenetic protein; cbEGF, : calcium-binding epidermal growth factor; ECM, : extracellular matrix; EGF, : epidermal growth factor; FUN, : fibrillin unique N-terminus; GDF, : growth and differentiation factor; HEK, : human embryonic kidney; HSPG, : heparan sulfate proteoglycan; hyb, : hybrid; LAP, : latency-associated propeptide; LTBP, : latent transforming growth factor-β-binding protein; MFS, : Marfan syndrome; PACE, : paired basic amino acid cleaving enzyme; TB, : TGFβ-binding protein-like; TGFβ, : transforming growth factor-β
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c terminal propeptide is required for fibrillin 1 secretion and blocks premature assembly through linkage to domains cbegf41 43
2014Co-Authors: Sacha A. Jensen, Georgia Aspinall, Penny A HandfordAbstract:Fibrillin Microfibrils are 10–12 nm diameter, extracellular matrix assemblies that provide dynamic tissues of metazoan species with many of their biomechanical properties as well as sequestering growth factors and cytokines. Assembly of fibrillin monomers into Microfibrils is thought to occur at the cell surface, with initial steps including proprotein processing, multimerization driven by the C terminus, and the head-to-tail alignment of adjacent molecules. At present the mechanisms that regulate Microfibril assembly are still to be elucidated. We have used structure-informed protein engineering to create a recombinant, GFP-tagged version of fibrillin-1 (GFP-Fbn) to study this process. Using HEK293T cells transiently transfected with GFP-Fbn constructs, we show that (i) the C-terminal propeptide is an essential requirement for the secretion of full-length fibrillin-1 from cells; (ii) failure to cleave off the C-terminal propeptide blocks the assembly of fibrillin-1 into Microfibrils produced by dermal fibroblasts; and (iii) the requirement of the propeptide for secretion is linked to the presence of domains cbEGF41-43, because either deletion or exchange of domains in this region leads to cellular retention. Collectively, these data suggest a mechanism in which the propeptide blocks a key site at the C terminus to prevent premature Microfibril assembly.
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Dissecting the fibrillin Microfibril: structural insights into organization and function.
2012Co-Authors: Sacha A. Jensen, Ian B. Robertson, Penny A HandfordAbstract:Force-bearing tissues such as blood vessels, lungs, and ligaments depend on the properties of elasticity and flexibility. The 10 to 12 nm diameter fibrillin Microfibrils play vital roles in maintaining the structural integrity of these highly dynamic tissues and in regulating extracellular growth factors. In humans, defective Microfibril function results in several diseases affecting the skin, cardiovascular, skeletal, and ocular systems. Despite the discovery of fibrillin-1 having occurred more than two decades ago, the structure and organization of fibrillin monomers within the Microfibrils are still controversial. Recent structural data have revealed strategies by which fibrillin is able to maintain its architecture in dynamic tissues without compromising its ability to interact with itself and other cell matrix components. This review summarizes our current knowledge of Microfibril structure, from individual fibrillin domains and the calcium-dependent tuning of pairwise interdomain interactions to Microfibril dynamics, and how this relates to Microfibril function in health and disease.
Sacha A. Jensen - One of the best experts on this subject based on the ideXlab platform.
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assembly assay identifies a critical region of human fibrillin 1 required for 10 12 nm diameter Microfibril biogenesis
2021Co-Authors: Sacha A. Jensen, Ondine Atwa, Penny A HandfordAbstract:The human FBN1 gene encodes fibrillin-1 (FBN1); the main component of the 10 - 12 nm diameter extracellular matrix Microfibrils. Marfan syndrome (MFS) is a common inherited connective tissue disorder, caused by FBN1 mutations. It features a wide spectrum of disease severity, from mild cases to the lethal neonatal form (nMFS), that is yet to be explained at the molecular level. Mutations associated with nMFS generally affect a region of FBN1 between domains TB3-cbEGF18 — the "neonatal region". To gain insight into the process of fibril assembly and increase our understanding of the mechanisms determining disease severity in MFS, we compared the secretion and assembly properties of FBN1 variants containing nMFS-associated substitutions with variants associated with milder, classical MFS (cMFS). In the majority of cases, both nMFS- and cMFS-associated neonatal region variants were secreted at levels comparable to wild type. Microfibril incorporation by the nMFS variants was greatly reduced or absent compared to the cMFS forms, however, suggesting that nMFS substitutions disrupt a previously undefined site of Microfibril assembly. Additional analysis of a domain deletion variant caused by exon skipping also indicates that register in the neonatal region is likely to be critical for assembly. These data demonstrate for the first time new requirements for Microfibril biogenesis and identify at least two distinct molecular mechanisms associated with disease substitutions in the TB3-cbEGF18 region; incorporation of mutant FBN1 into Microfibrils changing their integral properties (cMFS) or the blocking of wild type FBN1 assembly by mutant molecules that prevents late-stage lateral assembly (nMFS).
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new insights into the structure assembly and biological roles of 10 12 nm connective tissue Microfibrils from fibrillin 1 studies
2016Co-Authors: Sacha A. Jensen, Penny A HandfordAbstract:The 10-12 nm diameter Microfibrils of the extracellular matrix (ECM) impart both structural and regulatory properties to load-bearing connective tissues. The main protein component is the calcium-dependent glycoprotein fibrillin, which assembles into Microfibrils at the cell surface in a highly regulated process involving specific proteolysis, multimerization and glycosaminoglycan interactions. In higher metazoans, Microfibrils act as a framework for elastin deposition and modification, resulting in the formation of elastic fibres, but they can also occur in elastin-free tissues where they perform structural roles. Fibrillin Microfibrils are further engaged in a number of cell matrix interactions such as with integrins, bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-β (TGFβ). Fibrillin-1 (FBN1) mutations are associated with a range of heritable connective disorders, including Marfan syndrome (MFS) and the acromelic dysplasias, suggesting that the roles of 10-12 nm diameter Microfibrils are pleiotropic. In recent years the use of molecular, cellular and whole-organism studies has revealed that the Microfibril is not just a structural component of the ECM, but through its network of cell and matrix interactions it can exert profound regulatory effects on cell function. In this review we assess what is known about the molecular properties of fibrillin that enable it to assemble into the 10-12 nm diameter Microfibril and perform such diverse roles.
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new insights into the structure assembly and biological roles of 10 12 nm connective tissue Microfibrils from fibrillin 1 studies
2016Co-Authors: Sacha A. Jensen, Penny A HandfordAbstract:The 10–12 nm diameter Microfibrils of the extracellular matrix (ECM) impart both structural and regulatory properties to load-bearing connective tissues. The main protein component is the calcium-dependent glycoprotein fibrillin, which assembles into Microfibrils at the cell surface in a highly regulated process involving specific proteolysis, multimerization and glycosaminoglycan interactions. In higher metazoans, Microfibrils act as a framework for elastin deposition and modification, resulting in the formation of elastic fibres, but they can also occur in elastin-free tissues where they perform structural roles. Fibrillin Microfibrils are further engaged in a number of cell matrix interactions such as with integrins, bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-β (TGFβ). Fibrillin-1 ( FBN1 ) mutations are associated with a range of heritable connective disorders, including Marfan syndrome (MFS) and the acromelic dysplasias, suggesting that the roles of 10–12 nm diameter Microfibrils are pleiotropic. In recent years the use of molecular, cellular and whole-organism studies has revealed that the Microfibril is not just a structural component of the ECM, but through its network of cell and matrix interactions it can exert profound regulatory effects on cell function. In this review we assess what is known about the molecular properties of fibrillin that enable it to assemble into the 10–12 nm diameter Microfibril and perform such diverse roles. * ADAMTS(L), : a disintegrin and metalloproteinase with thrombospondin motifs(-like); BMP, : bone morphogenetic protein; cbEGF, : calcium-binding epidermal growth factor; ECM, : extracellular matrix; EGF, : epidermal growth factor; FUN, : fibrillin unique N-terminus; GDF, : growth and differentiation factor; HEK, : human embryonic kidney; HSPG, : heparan sulfate proteoglycan; hyb, : hybrid; LAP, : latency-associated propeptide; LTBP, : latent transforming growth factor-β-binding protein; MFS, : Marfan syndrome; PACE, : paired basic amino acid cleaving enzyme; TB, : TGFβ-binding protein-like; TGFβ, : transforming growth factor-β
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c terminal propeptide is required for fibrillin 1 secretion and blocks premature assembly through linkage to domains cbegf41 43
2014Co-Authors: Sacha A. Jensen, Georgia Aspinall, Penny A HandfordAbstract:Fibrillin Microfibrils are 10–12 nm diameter, extracellular matrix assemblies that provide dynamic tissues of metazoan species with many of their biomechanical properties as well as sequestering growth factors and cytokines. Assembly of fibrillin monomers into Microfibrils is thought to occur at the cell surface, with initial steps including proprotein processing, multimerization driven by the C terminus, and the head-to-tail alignment of adjacent molecules. At present the mechanisms that regulate Microfibril assembly are still to be elucidated. We have used structure-informed protein engineering to create a recombinant, GFP-tagged version of fibrillin-1 (GFP-Fbn) to study this process. Using HEK293T cells transiently transfected with GFP-Fbn constructs, we show that (i) the C-terminal propeptide is an essential requirement for the secretion of full-length fibrillin-1 from cells; (ii) failure to cleave off the C-terminal propeptide blocks the assembly of fibrillin-1 into Microfibrils produced by dermal fibroblasts; and (iii) the requirement of the propeptide for secretion is linked to the presence of domains cbEGF41-43, because either deletion or exchange of domains in this region leads to cellular retention. Collectively, these data suggest a mechanism in which the propeptide blocks a key site at the C terminus to prevent premature Microfibril assembly.
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Dissecting the fibrillin Microfibril: structural insights into organization and function.
2012Co-Authors: Sacha A. Jensen, Ian B. Robertson, Penny A HandfordAbstract:Force-bearing tissues such as blood vessels, lungs, and ligaments depend on the properties of elasticity and flexibility. The 10 to 12 nm diameter fibrillin Microfibrils play vital roles in maintaining the structural integrity of these highly dynamic tissues and in regulating extracellular growth factors. In humans, defective Microfibril function results in several diseases affecting the skin, cardiovascular, skeletal, and ocular systems. Despite the discovery of fibrillin-1 having occurred more than two decades ago, the structure and organization of fibrillin monomers within the Microfibrils are still controversial. Recent structural data have revealed strategies by which fibrillin is able to maintain its architecture in dynamic tissues without compromising its ability to interact with itself and other cell matrix components. This review summarizes our current knowledge of Microfibril structure, from individual fibrillin domains and the calcium-dependent tuning of pairwise interdomain interactions to Microfibril dynamics, and how this relates to Microfibril function in health and disease.
Michael J. Sherratt - One of the best experts on this subject based on the ideXlab platform.
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selective proteolysis by matrix metalloproteinases of photo oxidised dermal extracellular matrix proteins
2019Co-Authors: Sarah A Hibbert, Rachel E B Watson, Christopher E M Griffiths, Neil K Gibbs, Michael J. SherrattAbstract:Abstract Photodamage in chronically sun-exposed skin manifests clinically as deep wrinkles and histologically as extensive remodelling of the dermal extracellular matrix (ECM) and in particular, the elastic fibre system. We have shown previously that loss of fibrillin Microfibrils, a key elastic fibre component, is a hallmark of early photodamage and that these ECM assemblies are susceptible in vitro to physiologically attainable doses of ultraviolet radiation (UVR). Here, we test the hypotheses that UVR-mediated photo-oxidation is the primary driver of fibrillin Microfibril and fibronectin degradation and that prior UVR exposure will enhance the subsequent proteolytic activity of UVR-upregulated matrix metalloproteinases (MMPs). We confirmed that UVB (280-315 nm) irradiation in vitro induced structural changes to both fibrillin Microfibrils and fibronectin and these changes were largely reactive oxygen species (ROS)-driven, with increased ROS lifetime (D2O) enhancing protein damage and depleted O2 conditions abrogating it. Furthermore, we show that although exposure to UVR alone increased Microfibril structural heterogeneity, exposure to purified MMPs (1, −3, −7 and − 9) alone had minimal effect on Microfibril bead-to-bead periodicity; however, Microfibril suspensions exposed to UVR and then MMPs were more structurally homogenous. In contrast, the susceptibly of fibronectin to proteases was unaffected by prior UVR exposure. These observations suggest that both direct photon absorption and indirect production of ROS are important mediators of ECM remodelling in photodamage. We also show that fibrillin Microfibrils are relatively resistant to proteolysis by MMPs −1, −3, −7 and − 9 but that these MMPs may selectively remove damaged Microfibril assemblies. These latter observations have implications for predicting the mechanisms of tissue remodelling and targeted repair.
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structural and compositional diversity of fibrillin Microfibrils in human tissues
2018Co-Authors: Alexander Eckersley, Suzanne M Pilkington, Ronan Ocualain, Kieran T Mellody, Clair Baldock, David C. Knight, Christopher E M Griffiths, Rachel E B Watson, Michael J. SherrattAbstract:Elastic fibers comprising fibrillin Microfibrils and elastin are present in many tissues, including the skin, lungs, and arteries, where they confer elasticity and resilience. Although fibrillin Microfibrils play distinct and tissue-specific functional roles, it is unclear whether their ultrastructure and composition differ between elastin-rich (skin) and elastin-poor (ciliary body and zonule) organs or after in vitro synthesis by cultured cells. Here, we used atomic force microscopy, which revealed that the bead morphology of fibrillin Microfibrils isolated from the human eye differs from those isolated from the skin. Using newly developed pre-MS preparation methods and LC-MS/MS, we detected tissue-specific regions of the fibrillin-1 primary structure that were differentially susceptible to proteolytic extraction. Comparing tissue- and culture-derived Microfibrils, we found that dermis- and dermal fibroblast–derived fibrillin Microfibrils differ in both bead morphology and periodicity and also exhibit regional differences in fibrillin-1 proteolytic susceptibility. In contrast, collagen VI Microfibrils from the same dermal or fibroblast samples were invariant in ultrastructure (periodicity) and protease susceptibility. Finally, we observed that skin- and eye-derived Microfibril suspensions were enriched in elastic fiber– and basement membrane–associated proteins, respectively. LC-MS/MS also identified proteins (such as calreticulin and protein-disulfide isomerase) that are potentially fundamental to fibrillin Microfibril biology, regardless of their tissue source. Fibrillin Microfibrils synthesized in cell culture lacked some of these key proteins (MFAP2 and -4 and fibrillin-2). These results showcase the structural diversity of these key extracellular matrix assemblies, which may relate to their distinct roles in the tissues where they reside.
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proteomic analysis of fibrillin rich Microfibrils
2006Co-Authors: Stuart A Cain, Stephen Ball, Amanda Morgan, Michael J. Sherratt, Cay M. KieltyAbstract:MS has been used to investigate the composition of fibrillin-rich Microfibrils from non-elastic and elastic tissues, and to compare fibrillin-1 tryptic fingerprints derived from whole zonules, Microfibrils and recombinant fibrillin-1. In all Microfibril preparations, fibrillin-1 was abundant and the only fibrillin isoform. MAGP-1 was the only other Microfibril-associated molecule. gamma-Crystallin co-purified with zonular Microfibrils, so this association may contribute to ciliary zonule anchorage to lens. Recombinant fibrillin-1 tryptic peptides mapped throughout the molecule and included virtually all predicted peptides except for those larger than 4.5 kDa, smaller than 600 Da or post-translationally modified. In contrast, fewer Microfibril tryptic fibrillin-1 peptides were detected, although they were derived from domains throughout the molecule and included two peptides after the C-terminal furin processing site. Several Microfibril-derived N- and C-terminal domains never yielded any peptides, while tryptic peptides from other domains yielded numerous peptides, suggesting that some tissue Microfibril features are retained after trypsinisation. This first MS analysis of a purified extracellular matrix assembly has provided new insights into Microfibril composition and fibrillin-1 organisation within them.
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fibulin 5 interacts with fibrillin 1 molecules and Microfibrils
2005Co-Authors: Lyle J Freeman, Nigel Hodson, Kieran T Mellody, Anthony S. Weiss, Amanda Lomas, Adrian Shuttleworth, Michael J. Sherratt, Cay M. KieltyAbstract:Fibulin-5 plays an important role in elastic fibre formation in vivo. We have investigated the molecular interactions between fibulin-5 and components of fibrillin-rich Microfibrils which form a template for elastin. Fibulin-5 interacted in a dose-dependent manner with a fibrillin-1 N-terminal sequence and with tropoelastin, but not with MAGP-1 (Microfibril-associated glycoprotein-1) or decorin. Fibulin-5 did not inhibit interactions between fibrillin-1 N- and C-terminal fragments, or fibrillin-1 interactions with tropoelastin. Fibulin-5 may provide a link between tropoelastin and Microfibrils in the pericellular space during elastic fibre assembly.
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fibrillin Microfibrils are stiff reinforcing fibres in compliant tissues
2003Co-Authors: Michael J. Sherratt, Louise J Haston, David F Holmes, Clair Baldock, Carolyn J P Jones, Timothy J. Wess, Cay M. KieltyAbstract:Fibrillin-rich Microfibrils have endowed tissues with elasticity throughout multicellular evolution. We have used molecular combing techniques to determine Young's modulus for individual Microfibrils and X-ray diffraction of zonular filaments of the eye to establish the linearity of Microfibril periodic extension. Microfibril periodicity is not altered at physiological zonular tissue extensions and Young's modulus is between 78 MPa and 96 MPa, which is two orders of magnitude stiffer than elastin. We conclude that elasticity in Microfibril-containing tissues arises primarily from reversible alterations in supra-Microfibrillar arrangements rather than from intrinsic elastic properties of individual Microfibrils which, instead, act as reinforcing fibres in fibrous composite tissues.
Daniel J. Cosgrove - One of the best experts on this subject based on the ideXlab platform.
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the shape of native plant cellulose Microfibrils
2018Co-Authors: James D. Kubicki, Daniel P. Oehme, Daisuke Sawada, Hugh Oneill, Hui Yang, Daniel J. CosgroveAbstract:Determining the shape of plant cellulose Microfibrils is critical for understanding plant cell wall molecular architecture and conversion of cellulose into biofuels. Only recently has it been determined that these cellulose Microfibrils are composed of 18 cellulose chains rather than 36 polymers arranged in a diamond-shaped pattern. This study uses density functional theory calculations to model three possible habits for the 18-chain Microfibril and compares the calculated energies, structures, 13C NMR chemical shifts and WAXS diffractograms of each to evaluate which shape is most probable. Each model is capable of reproducing experimentally-observed data to some extent, but based on relative theoretical energies and reasonable reproduction of all variables considered, a Microfibril based on 5 layers in a 34443 arrangement is predicted to be the most probable. A habit based on a 234432 arrangement is slightly less favored, and a 6 × 3 arrangement is considered improbable.
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xyloglucan in the primary cell wall assessment by fesem selective enzyme digestions and nanogold affinity tags
2018Co-Authors: Yunzhen Zheng, Yuning Chen, Edward R Wagner, Xuan Wang, Daniel J. CosgroveAbstract:Summary Xyloglucan has been hypothesized to bind extensively to cellulose Microfibril surfaces and to tether Microfibrils into a load-bearing network, thereby playing a central role in wall mechanics and growth, but this view is challenged by newer results. Here we combined high-resolution imaging by field emission scanning electron microscopy (FESEM) with nanogold affinity tags and selective endoglucanase treatments to assess the spatial location and conformation of xyloglucan in onion cell walls. FESEM imaging of xyloglucanase-digested cell walls revealed an altered Microfibril organization but did not yield clear evidence of xyloglucan conformations. Backscattered electron detection provided excellent detection of nanogold affinity tags in the context of wall fibrillar organization. Labelling with xyloglucan-specific CBM76 conjugated with nanogold showed that xyloglucans were associated with fibril surfaces in both extended and coiled conformations, but tethered configurations were not observed. Labelling with nanogold-conjugated CBM3, which binds the hydrophobic surface of crystalline cellulose, was infrequent until the wall was predigested with xyloglucanase, whereupon Microfibril labelling was extensive. When tamarind xyloglucan was allowed to bind to xyloglucan-depleted onion walls, CBM76 labelling gave positive evidence for xyloglucans in both extended and coiled conformations, yet xyloglucan chains were not directly visible by FESEM. These results indicate that an appreciable, but still small, surface of cellulose Microfibrils in the onion wall is tightly bound with extended xyloglucan chains and that some of the xyloglucan has a coiled conformation.
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nanoscale movements of cellulose Microfibrils in primary cell walls
2017Co-Authors: Tian Zhang, Dimitrios Vavylonis, Daniel M Durachko, Daniel J. CosgroveAbstract:The growing plant cell wall is commonly considered to be a fibre-reinforced structure whose strength, extensibility and anisotropy depend on the orientation of crystalline cellulose Microfibrils, their bonding to the polysaccharide matrix and matrix viscoelasticity1–4. Structural reinforcement of the wall by stiff cellulose Microfibrils is central to contemporary models of plant growth, mechanics and meristem dynamics4–12. Although passive Microfibril reorientation during wall extension has been inferred from theory and from bulk measurements13–15, nanometre-scale movements of individual Microfibrils have not been directly observed. Here we combined nanometre-scale imaging of wet cell walls by atomic force microscopy (AFM) with a stretching device and endoglucanase treatment that induces wall stress relaxation and creep, mimicking wall behaviours during cell growth. Microfibril movements during forced mechanical extensions differ from those during creep of the enzymatically loosened wall. In addition to passive angular reorientation, we observed a diverse repertoire of Microfibril movements that reveal the spatial scale of molecular connections between Microfibrils. Our results show that wall loosening alters Microfibril connectivity, enabling Microfibril dynamics not seen during mechanical stretch. These insights into Microfibril movements and connectivities need to be incorporated into refined models of plant cell wall structure, growth and morphogenesis. Plant cell growth requires cell wall extension. Here, the nanoscale movement of cellulose Microfibrils in onion primary cell wall is imaged by atomic force microscopy and compared under mechanical extension versus enzymatic loosening.
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nanoscale movements of cellulose Microfibrils in primary cell walls
2017Co-Authors: Tian Zhang, Dimitrios Vavylonis, Daniel M Durachko, Daniel J. CosgroveAbstract:The growing plant cell wall is commonly considered to be a fibre-reinforced structure whose strength, extensibility and anisotropy depend on the orientation of crystalline cellulose Microfibrils, their bonding to the polysaccharide matrix and matrix viscoelasticity1-4. Structural reinforcement of the wall by stiff cellulose Microfibrils is central to contemporary models of plant growth, mechanics and meristem dynamics4-12. Although passive Microfibril reorientation during wall extension has been inferred from theory and from bulk measurements13-15, nanometre-scale movements of individual Microfibrils have not been directly observed. Here we combined nanometre-scale imaging of wet cell walls by atomic force microscopy (AFM) with a stretching device and endoglucanase treatment that induces wall stress relaxation and creep, mimicking wall behaviours during cell growth. Microfibril movements during forced mechanical extensions differ from those during creep of the enzymatically loosened wall. In addition to passive angular reorientation, we observed a diverse repertoire of Microfibril movements that reveal the spatial scale of molecular connections between Microfibrils. Our results show that wall loosening alters Microfibril connectivity, enabling Microfibril dynamics not seen during mechanical stretch. These insights into Microfibril movements and connectivities need to be incorporated into refined models of plant cell wall structure, growth and morphogenesis.
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spatial organization of cellulose Microfibrils and matrix polysaccharides in primary plant cell walls as imaged by multichannel atomic force microscopy
2016Co-Authors: Tian Zhang, Yunzhen Zheng, Daniel J. CosgroveAbstract:Summary We used atomic force microscopy (AFM), complemented with electron microscopy, to characterize the nanoscale and mesoscale structure of the outer (periclinal) cell wall of onion scale epidermis – a model system for relating wall structure to cell wall mechanics. The epidermal wall contains ~100 lamellae, each ~40 nm thick, containing 3.5-nm wide cellulose Microfibrils oriented in a common direction within a lamella but varying by ~30 to 90° between adjacent lamellae. The wall thus has a crossed polylamellate, not helicoidal, wall structure. Montages of high-resolution AFM images of the newly deposited wall surface showed that single Microfibrils merge into and out of short regions of Microfibril bundles, thereby forming a reticulated network. Microfibril direction within a lamella did not change gradually or abruptly across the whole face of the cell, indicating continuity of the lamella across the outer wall. A layer of pectin at the wall surface obscured the underlying cellulose Microfibrils when imaged by FESEM, but not by AFM. The AFM thus preferentially detects cellulose Microfibrils by probing through the soft matrix in these hydrated walls. AFM-based nanomechanical maps revealed significant heterogeneity in cell wall stiffness and adhesiveness at the nm scale. By color coding and merging these maps, the spatial distribution of soft and rigid matrix polymers could be visualized in the context of the stiffer Microfibrils. Without chemical extraction and dehydration, our results provide multiscale structural details of the primary cell wall in its near-native state, with implications for Microfibrils motions in different lamellae during uniaxial and biaxial extensions.
