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Fibronectin assembly

Clifford M Csizmar, Jacob Petersburg, Alexander Hendricks, Lawrence A Stern, Benjamin J Hackel, Carston R Wagner
Membrane-engineered cells displaying antigen-targeting ligands are useful as both scientific tools and clinical therapeutics. While genetically-encoded artificial receptors have proven efficacious, their scope remains limited as this approach is not amenable to all cell types and the modification is often permanent. Our group has developed a non-genetic method to rapidly, stably, and reversibly modify any cell membrane with a chemically self-assembled nanoring (CSAN) that can function as a prosthetic receptor...
March 14, 2018: Bioconjugate Chemistry
Yu Bin Lee, Eun Mi Kim, Hayeon Byun, Hyung-Kwan Chang, Kwanghee Jeong, Zachary M Aman, Yu Suk Choi, Jungyul Park, Heungsoo Shin
Numerous methods have been reported for the fabrication of 3D multi-cellular spheroids and their use in stem cell culture. Current methods typically relying on the self-assembly of trypsinized, suspended stem cells, however, show limitations with respect to cell viability, throughput, and accurate recapitulation of the natural microenvironment. In this study, we developed a new system for engineering cell spheroids by self-assembly of micro-scale monolayer of stem cells. We prepared synthetic hydrogels with the surface of chemically formed micropatterns (squares/circles with width/diameter of 200 μm) on which mesenchymal stem cells isolated from human nasal turbinate tissue (hTMSCs) were selectively attached and formed a monolayer...
March 1, 2018: Biomaterials
Albert Lee, Dimitrios Karamichos, Obianamma E Onochie, Audrey E K Hutcheon, Celeste B Rich, James D Zieske, Vickery Trinkaus-Randall
Deposition of matrix proteins during development and repair is critical to the transparency of the cornea. While many cells respond to a hypoxic state that can occur in a tumor, the cornea is exposed to hypoxia during development prior to eyelid opening and during the diurnal sleep cycle where oxygen levels can drop from 21% to 8%. In this study, we used 2 three-dimensional (3-D) models to examine how stromal cells respond to periods of acute hypoxic states. The first model, a stromal construct model, is a 3-D stroma-like construct that consists of human corneal fibroblasts (HCFs) stimulated by a stable form of ascorbate for 1, 2, and 4 weeks to self-assemble their own extracellular matrix...
February 26, 2018: Experimental Eye Research
Abshar Hasan, Varun Saxena, Lalit M Pandey
Although metallic biomaterials find numerous biomedical applications, their inherent low bioactivity and poor osteointegration had been a great challenge since decades. Surface modification via silanization can serve as an attractive method for improving aforementioned properties of such substrates. However, its effect on protein adsorption/conformation and subsequent cell adhesion and spreading, has rarely been investigated. This work reports the in-depth study of the effect of Ti6Al4V surface functionalization on protein adsorption and cell behaviour...
February 28, 2018: Langmuir: the ACS Journal of Surfaces and Colloids
Huong Nguyen, Khon Huynh, Volker R Stoldt
Soluble plasma fibronectin (Fn) with its inactive compact structure requires unfolding to assemble into active fibrils, which play a role in hemostasis and thrombosis. Fn fibril assembly involves Fn binding to cell receptors, biomechanical coupling of Fn to the cytoskeleton by integrins, exposure of self-assembly sites via contractile cell forces, and elongation of fibrils by Fn polymerization. In this report, we investigated the effect of platelet integrins and actin cytoskeleton on conformational changes of Fn induced by shear...
February 19, 2018: Biochemical and Biophysical Research Communications
Moonyoung Lee, Eunyoung Lee, Sun Hee Jin, Sungjin Ahn, Sae On Kim, Jungmin Kim, Dalwoong Choi, Kyung-Min Lim, Seung-Taek Lee, Minsoo Noh
The role of leptin in cutaneous wound healing process has been suggested in genetically obese mouse studies. However, the molecular and cellular effects of leptin on human epidermal keratinocytes are still unclear. In this study, the whole-genome-scale microarray analysis was performed to elucidate the effect of leptin on epidermal keratinocyte functions. In the leptin-treated normal human keratinocytes (NHKs), we identified the 151 upregulated and 53 downregulated differentially expressed genes (DEGs). The gene ontology (GO) enrichment analysis with the leptin-induced DEGs suggests that leptin regulates NHKs to promote pro-inflammatory responses, extracellular matrix organization, and angiogenesis...
February 21, 2018: Archives of Dermatological Research
Yongbo Lu, Suzan A Kamel-El Sayed, Kun Wang, LeAnn M Tiede-Lewis, Michael A Grillo, Patricia A Veno, Vladimir Dusevich, Charlotte L Phillips, Lynda F Bonewald, Sarah L Dallas
Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry...
February 20, 2018: Journal of Bone and Mineral Research: the Official Journal of the American Society for Bone and Mineral Research
Mohan Nair, Juan Romero, Aria Mahtabfar, Ahmed M Meleis, Ramsey A Foty, Siobhan A Corbett
Dispersal of Glioblastoma (GBM) renders localized therapy ineffective and is a major cause of recurrence. Previous studies have demonstrated that Dexamethasone (Dex), a drug currently used to treat brain tumor-related edema, can also significantly reduce dispersal of human primary GBM cells from neurospheres. It does so by triggering α5 integrin activity, leading to restoration of fibronectin matrix assembly (FNMA), increased neurosphere cohesion, and reduction of neurosphere dispersal velocity (DV). How Dex specifically activates α5 integrin in these GBM lines is unknown...
February 14, 2018: International Journal of Molecular Sciences
Aicha Melouane, Antoine Carbonell, Mayumi Yoshioka, Jack Puymirat, Jonny St-Amand
Secreted protein, acidic and rich in cysteine (SPARC) is differentially associated with cell proliferation and extracellular matrix (ECM) assembly. We show here the effect of exogenous SPARC inhibition/induction on ECM and mitochondrial proteins expression and on the differentiation of C2C12 cells. The cells were cultured in growth medium (GM) supplemented with different experimental conditions. The differentiation of myoblasts was studied for 5 days, the expressions of ECM and mitochondrial proteins were measured and the formation of the myotubes was quantified after exogenous induction/inhibition of SPARC...
2018: PloS One
Qin Wang, Julie Delcorde, Tracy Tang, Gregory P Downey, Christopher A McCulloch
IL-1 signaling is adhesion-restricted in many cell types, but the mechanism that drives it is not defined. We screened for proteins recruited to nascent adhesions in IL-1-treated human fibroblasts with tandem mass tag-mass spectrometry. We used fibronectin bead preparations to enrich 10 actin-associated proteins. There was a 1.2 times log 2-fold enrichment of actin capping protein (ACP) at 30 min after IL-1 stimulation. Knockdown (KD) of ACP by siRNA reduced IL-1-induced ERK activation(by 56%, matrix metalloproteinase-3 (MMP-3) expression by 48%, and MMP-9 expression by 62% (in all reductions, P < 0...
January 22, 2018: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
Seungkuk Ahn, Keel Yong Lee, Kevin Kit Parker, Kwanwoo Shin
The extracellular matrix (ECM) consists of polymerized protein monomers that form a unique fibrous network providing stability and structural support to surrounding cells. We harnessed the fibrillogenesis mechanisms of naturally occurring ECM proteins to produce artificial fibers with a heterogeneous protein makeup. Using ECM proteins as fibril building blocks, we created uniquely structured multi-component ECM fibers. Sequential incubation of fibronectin (FN) and laminin (LAM) resulted in self-assembly into locally stacked fibers...
January 30, 2018: Scientific Reports
Bianca R Tomasini-Johansson, Deane F Mosher
Fibronectin (FN) is a plasma glycoprotein produced by hepatocytes that circulates at near micromolar concentration and assembles into extracellular matrix fibrils at cell surfaces along with locally produced cellular FN. We describe two microplate assays that quantify assembly of human FN by cells in monolayer culture. One assay measures fluorescence due to incorporation of ALEXA488-plasma FN into matrices of fibroblasts and has been used successfully in high-throughput screens. The other measures fluorescence due to binding of fluorochrome-labeled antibody to the EDA domain of cellular FN synthesized and deposited by various cell types...
2018: Methods in Cell Biology
David Kimelman, Natalie L Smith, Jason Kuan Han Lai, Didier Yr Stainier
The vertebrate embryo undergoes a series of dramatic morphological changes as the body extends to form the complete anterior-posterior axis during the somite-forming stages. The molecular mechanisms regulating these complex processes are still largely unknown. We show that the Hippo pathway transcriptional coactivators Yap1 and Wwtr1 are specifically localized to the presumptive epidermis and notochord, and play a critical and unexpected role in posterior body extension by regulating Fibronectin assembly underneath the presumptive epidermis and surrounding the notochord...
December 28, 2017: ELife
Soichi Iwai, Satoko Kishimoto, Yuto Amano, Akihiro Nishiguchi, Michiya Matsusaki, Akinori Takeshita, Mitsuru Akashi
Preventing cancer metastasis requires a thorough understanding of cancer cell invasion. These phenomena occur in human 3D living tissues. To this end, we developed a human cell-based three-dimensional (3D) cultured tissue constructs that imitate in vivo human tissue organization. We investigated whether our 3D-cell culture system can be used to analyze the invasion of human oral squamous cell carcinoma (OSCC) cells. The 3D-tissue structure consisted of five layers of normal human dermal fibroblasts along with human dermal lymphatic endothelial cell tubes and was generated by the cell accumulation technique and layer-by-layer assembly using fibronectin and gelatin...
December 27, 2017: Journal of Biomedical Materials Research. Part A
Christopher A Lemmon, Seth H Weinberg
The mechanism of assembly of the extracellular matrix protein fibronectin (FN) into elastic, insoluble fibrils is still poorly understood. FN fibrillogenesis requires cell-generated forces, which expose cryptic FN-FN binding sites buried in FN Type III domains. The number and location of cryptic binding sites have been debated, but experimental evidence suggests multiple domains may contain FN-FN binding sites. The requirement of cell-dependent forces to generate FN fibrils restricts investigation of the mechanism of assembly...
December 22, 2017: Scientific Reports
Narayanan Madaboosi, Katja Uhlig, Stephan Schmidt, Anna S Vikulina, Helmuth Möhwald, Claus Duschl, Dmitry Volodkin
Polyelectrolyte multilayers assembled from hyaluronic acid (HA) and poly-l-lysine (PLL) are most widely studied showing excellent reservoir characteristics to host molecules of diverse nature; however, thick (HA/PLL)n films are often found cell repellent. By a systematic study of the adhesion and proliferation of various cells as a function of bilayer number "n" a correlation with the mechanical and chemical properties of films is developed. The following cell lines have been studied: mouse 3T3 and L929 fibroblasts, human foreskin primary fibroblasts VH-Fib, human embryonic kidney HEK-293, human bone cell line U-2-OS, Chinese hamster ovary CHO-K and mouse embryonic stem cells...
February 2018: Macromolecular Bioscience
Irene Raitman, Mia L Huang, Selwyn A Williams, Benjamin Friedman, Kamil Godula, Jean E Schwarzbauer
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...
December 6, 2017: Matrix Biology: Journal of the International Society for Matrix Biology
Hendrik Deschout, Ilia Platzman, Daniel Sage, Lely Feletti, Joachim P Spatz, Aleksandra Radenovic
Cells rely on focal adhesions (FAs) to carry out a variety of important tasks, including motion, environmental sensing, and adhesion to the extracellular matrix. Although attaining a fundamental characterization of FAs is a compelling goal, their extensive complexity and small size, which can be below the diffraction limit, have hindered a full understanding. In this study we have used single-molecule localization microscopy (SMLM) to investigate integrin β3 and paxillin in rat embryonic fibroblasts growing on two different extracellular matrix-representing substrates (i...
December 5, 2017: Biophysical Journal
Ekaterina Kalabusheva, Vasily Terskikh, Ekaterina Vorotelyak
Hair follicle (HF) reconstruction in vitro is a promising field in alopecia treatment and human HF development research. Here, we combined postnatal human dermal papilla (DP) cells and skin epidermal keratinocytes (KCs) in a hanging drop culture to develop an artificial HF germ. The method is based on DP cell hair-inducing properties and KC self-organization. We evaluated two protocols of aggregate assembling. Mixed HF germ-like structures demonstrated the initiation of epithelial-mesenchymal interaction, including WNT pathway activation and expression of follicular markers...
2017: Stem Cells International
Audrey Gossart, Kyle G Battiston, Adeline Gand, Emmanuel Pauthe, J Paul Santerre
Monocyte interactions with materials that are biofunctionalized with fibronectin (Fn) are of interest because of the documented literature which associates this protein with white blood cell function at implant sites. A degradable-polar hydrophobic ionic polyurethane (D-PHI), has been reported to promote an anti-inflammatory response from human monocytes. The aim of the current work was to study the influence of intrinsic D-PHI material chemistry on Fn adsorption (mono and multi-layer structures), and to investigate the influence of such chemistry on the structural state of the Fn, as well as the latter's influence on the activity of human monocytes on the protein coated substrates...
January 15, 2018: Acta Biomaterialia
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