Collective cell migration on artificial extracellular matrix proteins containing full-length fibronectin domains.
Ontology highlight
ABSTRACT:
SUBMITTER: Fong E
PROVIDER: S-EPMC3027490 | biostudies-literature | 2010 Dec
REPOSITORIES: biostudies-literature
Publications
Collective cell migration on artificial extracellular matrix proteins containing full-length fibronectin domains.
Fong Eileen E Tirrell David A DA
Advanced materials (Deerfield Beach, Fla.) 20101201 46
Similar Datasets
Project description:In the process of matrix assembly, multivalent extracellular matrix (ECM) proteins are induced to self-associate and to interact with other ECM proteins to form fibrillar networks. Matrix assembly is usually initiated by ECM glycoproteins binding to cell surface receptors, such as fibronectin (FN) dimers binding to ?5ß1 integrin. Receptor binding stimulates FN self-association mediated by the N-terminal assembly domain and organizes the actin cytoskeleton to promote cell contractility. FN conformational changes expose additional binding sites that participate in fibril formation and in conversion of fibrils into a stabilized, insoluble form. Once assembled, the FN matrix impacts tissue organization by contributing to the assembly of other ECM proteins. Here, we describe the major steps, molecular interactions, and cellular mechanisms involved in assembling FN dimers into fibrillar matrix while highlighting important issues and major questions that require further investigation.
| S-EPMC3628685 | biostudies-literature
Project description:During zebrafish gastrulation the planar cell polarity (PCP) protein Vang-like 2 (Vangl2) regulates the polarization of cells that are engaged in directed migration. However, it is unclear whether Vangl2 influences membrane-protrusive activities in migrating gastrula cells and whether these processes require the fibronectin extracellular matrix. Here, we report that Vangl2 modulates the formation and polarization of actin-rich filopodia-like and large lamellipodia-like protrusions in ectodermal cells. By contrast, disrupted Glypican4/PCP signaling affects protrusion polarity but not protrusion number or directed migration. Analysis of fluorescent fusion protein expression suggests that there is widespread Vangl2 symmetry in migrating cells, but there is enrichment at membrane domains that are developing large protrusions compared with non-protrusive domains. We show that the fibronectin extracellular matrix is essential for cell-surface Vangl2 expression, membrane-protrusive activity and directed migration. Manipulation of fibronectin protein levels rescues protrusion and directed migration phenotypes in vangl2 mutant embryos, but it is not sufficient to restore either PCP, or convergence and extension movements. Together, our findings identify distinct roles for Vangl2 and Glypican4/PCP signaling during membrane protrusion formation and demonstrate that cell-matrix interactions underlie Vangl2-dependent regulation of protrusive activities in migrating gastrula cells.
| S-EPMC6262793 | biostudies-literature
Project description:The extracellular matrix is essential for tissue formation and regeneration through the control of cellular behavior. Deregulation of extracellular matrix components is associated to disease, including neurodegeneration. After peripheral nerve injury, Schwann cells guide regrowing axons to their targets. These glial cells migrate collectively and provide an extracellular environment to enable neural repair. How this occurs remains poorly understood. Here, we show that Sox2 controls fibronectin fibrillogenesis in Schwann cells to provide a highly oriented extracellular matrix, which supports their rapid collective migration with a continuous cellular flow. Sox2 directly activates fibronectin expression in Schwann cells, leading to an increase in fibrillogenesis and cellular huddling. Accordingly, loss of fibrillogenesis leads to glial disassembly and disorganized axon regrowth. In vivo, 7 days post nerve injury, we found that pro-regenerative Schwann cells co-express Sox2 and the EIIIA-containing fibronectin splicing isoform. This mechanism is conserved in mammals, including humans, but absent in zebrafish. Taken together, our results demonstrate that Sox2 directly controls fibrillogenesis and provide a novel mechanism for the modification of the environmental architecture by glial cells during neuronal repair.
2020-02-11 | GSE94590 | GEO
Project description:Collective cell migration is involved in many biological and pathological processes. Various factors have been shown to regulate the decision to migrate collectively or individually, but the impact of cell-extracellular matrix (ECM) interactions is still debated. Here, we developed a method for analyzing collective cell migration by precisely tuning the interactions between cells and ECM ligands. Gold nanoparticles are arrayed on a glass substrate with a defined nanometer spacing by block copolymer micellar nanolithography (BCML), and photocleavable poly(ethylene glycol) (Mw ?=? 12 kDa, PEG12K) and a cyclic RGD peptide, as an ECM ligand, are immobilized on this substrate. The remaining glass regions are passivated with PEG2K-silane to make cells interact with the surface via the nanoperiodically presented cyclic RGD ligands upon the photocleavage of PEG12K. On this nanostructured substrate, HeLa cells are first patterned in photo-illuminated regions, and cell migration is induced by a second photocleavage of the surrounding PEG12K. The HeLa cells gradually lose their cell-cell contacts and become disconnected on the nanopatterned substrate with 10-nm particles and 57-nm spacing, in contrast to their behavior on the homogenous substrate. Interestingly, the relationship between the observed migration collectivity and the cell-ECM ligand interactions is the opposite of that expected based on conventional soft matter models. It is likely that the reduced phosphorylation at tyrosine-861 of focal adhesion kinase (FAK) on the nanopatterned surface is responsible for this unique migration behavior. These results demonstrate the usefulness of the presented method in understanding the process of determining collective and non-collective migration features in defined micro- and nano-environments and resolving the crosstalk between cell-cell and cell-ECM adhesions.
| S-EPMC3954836 | biostudies-literature
Project description:During extracellular matrix (ECM) assembly, fibronectin (FN) fibrils are irreversibly converted into a detergent-insoluble form which, through FN's multi-domain structure, can interact with collagens, matricellular proteins, and growth factors to build a definitive matrix. FN also has heparin/heparan sulfate (HS) binding sites. Using HS-deficient CHO cells, we show that the addition of soluble heparin significantly increased the amount of FN matrix that these cells assemble. Sulfated HS glycosaminoglycan (GAG) mimetics similarly increased FN assembly and demonstrated a dependence on GAG sulfation. The length of the heparin chains also plays a role in assembly. Chains of sufficient length to bind to two FN molecules gave maximal stimulation of assembly whereas shorter heparin had less of an effect. Using a decellularized fibroblast matrix for proteolysis, detergent fractionation, and mass spectrometry, we found that the predominant domain within insoluble fibril fragments is FN's major heparin-binding domain HepII (modules III12-14). Multiple HepII domains bind simultaneously to a single heparin chain in size exclusion chromatography analyses. We propose a model in which heparin/HS binding to the HepII domain connects multiple FNs together to facilitate the formation of protein interactions for insoluble fibril assembly.
| S-EPMC5910196 | biostudies-literature
Project description:The extracellular matrix (ECM) has been demonstrated to facilitate angiogenesis. In particular, fibronectin has been documented to activate endothelial cells, resulting in their transition from a quiescent state to an active state in which the cells exhibit enhanced migration and proliferation. The goal of this study is to examine the role of polymerized fibronectin during vascular tubulogenesis using a 3 dimensional (3D) cell-derived de-cellularized matrix. A fibronectin-rich 3D de-cellularized ECM was used as a scaffold to study vascular morphogenesis of endothelial cells (ECs). Confocal analyses of several matrix proteins reveal high intra- and extra-cellular deposition of fibronectin in formed vascular structures. Using a small peptide inhibitor of fibronectin polymerization, we demonstrate that inhibition of fibronectin fibrillogenesis in ECs cultured atop de-cellularized ECM resulted in decreased vascular morphogenesis. Further, immunofluorescence and ultrastructural analyses reveal decreased expression of stromal matrix proteins in the absence of polymerized fibronectin with high co-localization of matrix proteins found in association with polymerized fibronectin. Evaluating vascular kinetics, live cell imaging showed that migration, migration velocity, and mean square displacement, are disrupted in structures grown in the absence of polymerized fibronectin. Additionally, vascular organization failed to occur in the absence of a polymerized fibronectin matrix. Consistent with these observations, we tested vascular morphogenesis following the disruption of EC adhesion to polymerized fibronectin, demonstrating that block of integrins α5β1 and αvβ3, abrogated vascular morphogenesis. Overall, fibronectin deposition in a 3D cell-derived de-cellularized ECM appears to be imperative for matrix assembly and vascular morphogenesis.
| S-EPMC4728102 | biostudies-literature
Project description:BackgroundInfectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria.ResultsA new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence.ConclusionsThese results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses.
| S-EPMC3840674 | biostudies-literature
Project description:Heat shock protein 90 (HSP90) is an evolutionarily conserved chaperone protein that controls the function and stability of a wide range of cellular client proteins. Fibronectin (FN) is an extracellular client protein of HSP90, and exogenous HSP90 or inhibitors of HSP90 alter the morphology of the extracellular matrix. Here, we further characterized the HSP90 and FN interaction. FN bound to the M domain of HSP90 and interacted with both the open and closed HSP90 conformations; and the interaction was reduced in the presence of sodium molybdate. HSP90 interacted with the N-terminal regions of FN, which are known to be important for matrix assembly. The highest affinity interaction was with the 30-kDa (heparin-binding) FN fragment, which also showed the greatest colocalization in cells and accommodated both HSP90 and heparin in the complex. The strength of interaction with HSP90 was influenced by the inherent stability of the FN fragments, together with the type of motif, where HSP90 preferentially bound the type-I FN repeat over the type-II repeat. Exogenous extracellular HSP90 led to increased incorporation of both full-length and 70-kDa fragments of FN into fibrils. Together, our data suggested that HSP90 may regulate FN matrix assembly through its interaction with N-terminal FN fragments.
| S-EPMC7072298 | biostudies-literature
Project description:The physical structure of the extracellular matrix (ECM) is tissue-specific and fundamental to normal tissue function. Proper alignment of ECM fibers is essential for the functioning of a variety of tissues. While matrix assembly in general has been intensively investigated, little is known about the mechanisms required for formation of aligned ECM fibrils. We investigated the initiation of fibronectin (FN) matrix assembly using fibroblasts that assemble parallel ECM fibrils and found that matrix assembly sites, where FN fibrillogenesis is initiated, were oriented in parallel at the cell poles. We show that these polarized matrix assembly sites progress into fibrillar adhesions and ultimately into aligned FN fibrils. Cells that assemble an unaligned meshwork matrix form matrix assembly sites around the cell periphery, but the distribution of matrix assembly sites in these cells could be modulated through micropatterning or mechanical stretch. While an elongated cell shape corresponds with a polarized matrix assembly site distribution, these two features are not absolutely linked, since we discovered that transforming growth factor beta (TGF-β1) enhances matrix assembly site polarity and assembly of aligned fibrils independent of cell elongation. We conclude that the ultimate orientation of FN fibrils is determined by the alignment and distribution of matrix assembly sites that form during the initial stages of cell-FN interactions.
| S-EPMC8108514 | biostudies-literature
Project description:Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90? was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90?. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis.
| S-EPMC3899338 | biostudies-literature