PhD student, Francis Crick Institute
Atomistic analysis into the adhesion of fibronectin which dictates the in vivo properties
Fibronectin (FN) is a large extracellular matrix glycoprotein that affects many cell processes including differentiation, migration and proliferation. FN’s function requires its quaternary structure to transition from a compact to an extended form; this process is integrin dependant and leads to FN network formation. Recently much research was carried out on an alternative to this integrin dependant process - a material driven approach, with the significant portion of the work carried out on substrates made of poly (methyl/ethyl) acrylates by Prof. Manuel Salmeron-Sanchez’s group in Glasgow. FN on poly(ethyl acrylate) assumes the extended form, leading to network assembly, whereas the chemically almost-identical surface poly(methyl acrylate) shows FN aggregation and no network formation. To gain further insight into how such a small difference in the surface chemistry has has such drastic consequences in FN’s structure, we used molecular dynamics to simulate the crucial domains FN III 9-10 on self-assembled monolayers (SAMs) that were functionalised using the previously mentioned polymer’s side chains. Our simulations indicate that a) water hydration plays an important role in the adhesion of the two domains by creating dense solvation layers around the functional groups, and b) the residues that are particularly important in the FN-integrin binding, the RGD motif and the PHSRN synergy region, do not have any significance on this adhesion. These results are consistent with the latest mutational analysis (unpublished).
Abstract: Fibronectin (FN) is a multidomain protein with the ability to bind simultaneously to cell surface receptors, collagen, proteoglycans, and other FN molecules. Many of these domains and interactions are also involved in the assembly of FN dimers into a multimeric fibrillar matrix. When, where, and how FN binds to its various partners must be controlled and coordinated during fibrillogenesis. Steps in the process of FN fibrillogenesis including FN self-association, receptor activities, and intracellular pathways have been under intense investigation for years. In this review, the domain organization of FN including the extra domains and variable region that are controlled by alternative splicing are described. We discuss how FN-FN and cell-FN interactions play essential roles in the initiation and progression of matrix assembly using complementary results from cell culture and embryonic model systems that have enhanced our understanding of this process.
Pub.: 18 May '11, Pinned: 28 Jun '17
Abstract: Tissue morphogenesis is a complex process whereby tissue structures self-assemble by the aggregate behaviors of independently acting cells responding to both intracellular and extracellular cues in their environment. During embryonic development, morphogenesis is particularly important for organizing cells into tissues, and although key regulatory events of this process are well studied in isolation, a number of important systems-level questions remain unanswered. This is due, in part, to a lack of integrative tools that enable the coupling of biological phenomena across spatial and temporal scales. Here, we present a new computational framework that integrates intracellular signaling information with multi-cell behaviors in the context of a spatially heterogeneous tissue environment.We have developed a computational simulation of mesendoderm migration in the Xenopus laevis explant model, which is a well studied biological model of tissue morphogenesis that recapitulates many features of this process during development in humans. The simulation couples, via a JAVA interface, an ordinary differential equation-based mass action kinetics model to compute intracellular Wnt/beta-catenin signaling with an agent-based model of mesendoderm migration across a fibronectin extracellular matrix substrate. The emergent cell behaviors in the simulation suggest the following properties of the system: maintaining the integrity of cell-to-cell contact signals is necessary for preventing fractionation of cells as they move, contact with the Fn substrate and the existence of a Fn gradient provides an extracellular feedback loop that governs migration speed, the incorporation of polarity signals is required for cells to migrate in the same direction, and a delicate balance of integrin and cadherin interactions is needed to reproduce experimentally observed migratory behaviors.Our computational framework couples two different spatial scales in biology: intracellular with multicellular. In our simulation, events at one scale have quantitative and dynamic impact on events at the other scale. This integration enables the testing and identification of key systems-level hypotheses regarding how signaling proteins affect overall tissue-level behavior during morphogenesis in an experimentally verifiable system. Applications of this approach extend to the study of tissue patterning processes that occur during adulthood and disease, such as tumorgenesis and atherogenesis.
Pub.: 24 Oct '07, Pinned: 28 Jun '17
Abstract: Fibronectin, a major component of the extracellular matrix is critical for processes of cell traction and cell motility. Whole-mount confocal imaging of the three-dimensional architecture of the extracellular matrix is used to describe dynamic assembly and remodeling of fibronectin fibrils during gastrulation and neurulation in the early frog embryo. As previously reported, fibrils first appear under the prospective ectoderm. We describe here the first evidence for regulated assembly of fibrils along the somitic mesoderm/endoderm boundary as well as at the notochord/somitic mesoderm boundary and clearing of fibrils from the dorsal and ventral surfaces of the notochord that occurs over the course of a few hours. As gastrulation proceeds, fibrils are restored to the dorsal surface of the notochord, where the notochord contacts the prospective floor plate. As the neural folds form, fibrils are again remodeled as deep neural plate cells move medially. The process of neural tube closure leaves a region of the ectoderm overlying the neural crest transiently bare of fibrils. Fibrils are assembled surrounding the dorsal surface of the neural tube as the neural tube lumen is restored.
Pub.: 02 Nov '04, Pinned: 28 Jun '17
Abstract: In this study we demonstrate that planar cell polarity signaling regulates morphogenesis in Xenopus embryos in part through the assembly of the fibronectin (FN) matrix. We outline a regulatory pathway that includes cadherin adhesion and signaling through Rac and Pak, culminating in actin reorganization, myosin contractility, and tissue tension, which, in turn, directs the correct spatiotemporal localization of FN into a fibrillar matrix. Increased mechanical tension promotes FN fibril assembly in the blastocoel roof (BCR), while reduced BCR tension inhibits matrix assembly. These data support a model for matrix assembly in tissues where cell-cell adhesions play an analogous role to the focal adhesions of cultured cells by transferring to integrins the tension required to direct FN fibril formation at cell surfaces.
Pub.: 18 Mar '09, Pinned: 28 Jun '17
Abstract: Cells sense and respond to the mechanical properties of their microenvironment. We investigated whether these properties affect the ability of cells to assemble a fibrillar fibronectin (FN) matrix. Analysis of matrix assembled by cells grown on FN-coated polyacrylamide gels of varying stiffnesses showed that rigid substrates stimulate FN matrix assembly and activation of focal adhesion kinase (FAK) compared with the level of assembly and FAK signaling on softer substrates. Stimulating integrins with Mn(2+) treatment increased FN assembly on softer gels, suggesting that integrin binding is deficient on soft substrates. Guanidine hydrochloride-induced extension of the substrate-bound FN rescued assembly on soft substrates to a degree similar to that of Mn(2+) treatment and increased activation of FAK along with the initiation of assembly at FN matrix assembly sites. In contrast, increasing actin-mediated cell contractility did not rescue FN matrix assembly on soft substrates. Thus, rigidity-dependent FN matrix assembly is determined by extracellular events, namely the engagement of FN by cells and the induction of FN conformational changes. Extensibility of FN in response to substrate stiffness may serve as a mechanosensing mechanism whereby cells use pericellular FN to probe the stiffness of their environment.
Pub.: 17 Apr '13, Pinned: 28 Jun '17
Abstract: Advanced glycation endproducts (AGEs) are a heterogeneous group of compounds that form via non-enzymatic glycation of proteins throughout our lifespan and at a higher rate in certain chronic diseases such as diabetes. AGEs contribute to the progression of fibrosis, in part by stimulating cellular pathways that affect gene expression. Long-lived ECM proteins are targets for non-enzymatic glycation but the question of whether the AGE-modified ECM leads to excess ECM accumulation and fibrosis remains unanswered. In this study, cellular changes due to AGE accretion in the ECM were investigated. Non-enzymatic glycation of proteins in a decellularized fibroblast ECM was achieved by incubating the ECM in a solution of methylglyoxal (MGO). Mass spectrometry of fibronectin (FN) isolated from the glycated matrix identified twenty-eight previously unidentified MGO-derived AGE modification sites including functional sites such as the RGD integrin-binding sequence. Mesangial cells grown on the glycated, decellularized matrix assembled increased amounts of FN matrix. Soluble AGE-modified bovine serum albumin (BSA) also stimulated FN matrix assembly and this effect was reduced by function-blocking antibodies against the receptor for AGE (RAGE). These results indicate that cells respond to AGEs by increasing matrix assembly and that RAGE is involved in this response. This raises the possibility that the accumulation of ECM during the progression of fibrosis may be enhanced by cell interactions with AGEs on a glycated ECM.
Pub.: 15 Jul '16, Pinned: 28 Jun '17
Abstract: Retinal fibrosis, characterized by dysregulation of extracellular matrix (ECM) protein deposition by retinal endothelial cells, pigment epithelial cells, and other resident cell-types, is a unifying feature of several common retinal diseases. Fibronectin is an early constituent of newly deposited ECM and serves as a template for assembly of other ECM proteins, including collagens. Under physiologic conditions, fibronectin is found in all layers of Bruch's membrane. Proliferative vitreoretinopathy (PVR), a complication of retinal surgery, is characterized by ECM accumulation. Among the earliest histologic manifestations of diabetic retinopathy (DR) is capillary basement membrane thickening, which occurs due to perturbations in ECM homeostasis. Neovascularization, the hallmark of late stage DR as well as exudative age-related macular degeneration (AMD), involves ECM assembly as a scaffold for the aberrant new vessel architecture. Rodent models of retinal injury demonstrate a key role for fibronectin in complications characteristic of PVR, including retinal detachment. In mouse models of DR, reducing fibronectin gene expression has been shown to arrest the accumulation of ECM in the capillary basement membrane. Alterations in matrix metalloproteinase activity thought to be important in the pathogenesis of AMD impact the turnover of fibronectin matrix as well as collagens. Growth factors involved in PVR, AMD, and DR, such as PDGF and TGFβ, are known to stimulate fibronectin matrix assembly. A deeper understanding of how pathologic ECM deposition contributes to disease progression may help to identify novel targets for therapeutic intervention.
Pub.: 01 Nov '16, Pinned: 28 Jun '17
Abstract: A major challenge of tissue engineering is to generate materials that combine bioactivity with stability in a form that captures the robust nature of native tissues. Here we describe a procedure to fabricate a novel hybrid extracellular matrix (ECM) - synthetic scaffold biomaterial by cell-mediated deposition of ECM within an electrospun fiber mat. Synthetic polymer fiber mats were fabricated using poly(desamino tyrosyl-tyrosine carbonate) (PDTEC) co-spun with poly(ethylene glycol) (PEG) used as a sacrificial polymer. PEG removal increased the overall mat porosity and produced a mat with a layered structure that could be peeled into separate sheets of about 50 μm in thickness. Individual layers had pore sizes and wettability that facilitated cell infiltration over the depth of the scaffold. Confocal microscopy showed the formation of a highly inter-penetrated network of cells, fibronectin fibrils, and synthetic fibers mimicking a complex ECM as observed within tissues. Decellularization did not perturb the structure of the matrix or the fiber mat. The resulting hybrid ECM-scaffold promoted cell adhesion and spreading and stimulated new ECM assembly by stem cells and tumor cells. These results identify a new technique for fabricating highly porous synthetic fibrous scaffolds and an approach to supplement them with natural biomimetic cues. This article is protected by copyright. All rights reserved.
Pub.: 04 Apr '17, Pinned: 28 Jun '17
Abstract: Cell migration is a fundamental process involved in a wide range of biological phenomena. However, how the underlying mechanisms that control migration are orchestrated is not fully understood. In this work, we explore the migratory characteristics of human fibroblasts using different organisations of fibronectin (FN) triggered by two chemically similar surfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA); cell migration is mediated via an intermediate layer of fibronectin (FN). FN is organised into nanonetworks upon simple adsorption on PEA whereas a globular conformation is observed on PMA. We studied cell speed over the course of 24 h and the morphology of focal adhesions in terms of area and length. Additionally, we analysed the amount of cell-secreted FN as well as FN remodelling. Velocity of human fibroblasts was found to exhibit a biphasic behaviour on PEA, whereas it remained fairly constant on PMA. FA analysis revealed more mature focal adhesions on PEA over time contrary to smaller FAs found on PMA. Finally, human fibroblasts seemed to remodel adsorbed FN more on PMA than on PEA. Overall, these results indicate that the cell-protein-material interface affects cell migratory behaviour. Analysis of FAs together with FN secretion and remodelling were associated with differences in cell velocity providing insights into the factors that can modulate cell motility.
Pub.: 15 Jun '17, Pinned: 28 Jun '17
Abstract: We report the case of a 50-year-old woman with nephrotic-range proteinuria and lobular glomerulopathy on kidney biopsy. Homogenous glomerular deposits were non-immune reactive, but immunofluorescence microscopy for fibronectin was strongly positive. Ultrastructurally, the deposits were granular with focal fibril formation, leading to a diagnosis of fibronectin glomerulopathy. Mutational analysis revealed a heterozygous missense mutation in fibronectin (leading to the tyrosine at amino acid 973 being replaced by cysteine [Y973C]), confirming the diagnosis. This mutation affects Hep-III, one of the heparin-binding domains of fibronectin, and results in functional abnormalities.
Pub.: 23 Jun '12, Pinned: 06 Jun '17
Abstract: Fibronectin glomerulopathy occurs between the second and fifth decades of life in most patients, and it is known to be slowly progressive with mild proteinuria leading to kidney failure. The case of a 78-year-old woman with a rapid course of nephrotic syndrome due to fibronectin glomerulopathy is reported. She had proteinuria that rapidly increased to 6.8 g/day in a month and microscopic haematuria. A renal biopsy specimen showed lobular glomerulopathy and membranoproliferative glomerulonephritis-like lesions on light microscopy. There was scanty staining for immunoglobulins and complement. Electron microscopy revealed granular deposits with fibril formation. Immunohistochemistry of the fibronectin showed intense staining in the mesangium and peripheral loop. Therefore, this case was diagnosed as fibronectin glomerulopathy. The kidney function was rapidly decreasing, necessitating haemodialysis 2 months after renal biopsy. It is important to consider fibronectin glomerulopathy in the differential diagnosis of nephrotic syndrome in older people.
Pub.: 01 Oct '13, Pinned: 06 Jun '17
Abstract: Glomerulopathy with fibronectin deposits (GFND) is a rare autosomal dominant disease characterized by massive fibronectin deposits, leading to end-stage renal failure. Although mutations within the heparin-binding domains of the fibronectin 1 gene (FN1) have been associated with GFND, no mutations have been reported within the integrin-binding domains.In this study, FN1 mutational analysis was conducted in 12 families with GFND. Biochemical and functional features of mutated proteins were examined using recombinant fibronectin fragments encompassing both the integrin- and heparin-binding domains.We report six FN1 mutations from 12 families with GFND, including five that are novel (p.Pro969Leu, p.Pro1472del, p.Trp1925Cys, p.Lys1953_Ile1961del, and p.Leu1974Pro). p.Pro1472del is localized in the integrin-binding domain of fibronectin, while the others are in heparin-binding domains. We detected p.Tyr973Cys, p.Pro1472del, and p.Leu1974Pro mutations in multiple families, and haplotype analysis implied that p.Pro1472del and p.Leu1974Pro are founder mutations. The protein encoded by the novel integrin-binding domain mutation p.Pro1472del showed decreased cell binding ability via the integrin-binding site. Most affected patients developed urine abnormalities during the first or second decade of life, and some mutation carriers were completely asymptomatic.This is the second large-scale analysis of GFND families and the first report of an integrin-binding domain mutation. These findings may help determine the pathogenesis of GFND.
Pub.: 09 Apr '16, Pinned: 06 Jun '17