Showing papers by "Jean E. Schwarzbauer published in 2020"

Stimulation of Fibronectin Matrix Assembly by Lysine Acetylation

Abstract: Diabetic nephropathy, a devastating consequence of diabetes mellitus, is characterized by the accumulation of extracellular matrix (ECM) that disrupts the kidney's filtration apparatus. Elevated glucose levels increase the deposition of a fibronectin (FN) matrix by mesangial cells, the primary matrix-producing cells of the kidney, and also increase acetyl-CoA leading to higher levels of lysine acetylation. Here, we investigated the connection between acetylation and the ECM and show that treatment of mesangial cells with deacetylase inhibitors increases both acetylation and FN matrix assembly compared to untreated cells. The matrix effects were linked to lysine 794 (K794) in the β1 integrin cytoplasmic domain based on studies of cells expressing acetylated (K794Q) and non-acetylated (K794R) mimetics. β1(K794Q) cells assembled significantly more FN matrix than wildtype β1 cells, while the non-acetylated β1(K794R) form was inactive. We show that mutation of K794 affects FN assembly by stimulating integrin-FN binding activity and cell contractility. Wildtype and β1(K794Q) cells but not β1(K794R) cells further increased their FN matrix when stimulated with deacetylase inhibitors indicating that increased acetylation on other proteins is required for maximum FN assembly. Thus, lysine acetylation provides a mechanism for glucose-induced fibrosis by up-regulation of FN matrix assembly.

New mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy

Abstract: Fibronectin (Fn1) is an essential ECM glycoprotein important for embryonic development and homeostasis. The functions of Fn1 in regulating cell fate decisions, morphogenesis and cellular responses to injury are intimately linked to the process of Fn1 fibrillogenesis. Therefore, understanding the mechanisms by which Fn1 proteins assemble into fibrils is necessary to gain insights into diverse functions of Fn1. Using CRISPR/Cas9 mutagenesis, we generated mice and cell lines wherein a sequence encoding a fluorescent protein (FP) was knocked into the Fn1 locus replacing the termination codon, resulting in the expression of Fn1-FP proteins subject to endogenous regulation. Live imaging and super-resolution microscopy revealed that Fn1 fibrils are not continuous fibers as was thought before, instead, they are comprised of a discontinuous array of small nanodomains. Live imaging showed that Fn1 nanodomains are mobile and that they become arranged into progressively longer linear arrays as they move toward the nucleus in parallel with the rearward actin flow. The organization of Fn1 nanodomains into linear fibrillar arrays but not the formation of Fn1 nanodomains is regulated by the interactions mediated by the Fn1 N-terminal assembly domain. The nanodomain architecture of Fn1 fibrils is observed in multiple contexts: in three-dimensional ECM in vivo, on substrata of different composition and stiffness, and is retained when the linkage of Fn1 fibrils to cells is disrupted. The modular assembly and structure of Fn1 fibrils bears important implications for mechanisms of ECM remodeling and signal transduction.

Elevated glucose alters global gene expression and tenascin-C alternative splicing in mesangial cells.

Abstract: Mesangial cells are the major extracellular matrix (ECM)-producing cells in the kidney glomerulus and, when exposed to elevated glucose levels, they up-regulate assembly of fibronectin (FN) and other ECM proteins. Increases in glucose concentration are known to alter gene expression; here we investigated the connection between increased ECM production and changes in gene expression in mesangial cells. Comparison of mesangial cells grown in normal or high glucose conditions by RNA-sequencing showed significant expression changes in over 6000 genes and, when grouped by KEGG pathway analysis, identified the ECM-receptor interaction and focal adhesion pathways among the top 5 upregulated pathways. Of note was the significant increase in expression of tenascin-C (TN-C), a known regulator of FN matrix assembly. Mouse TN-C has multiple isoforms due to alternative splicing of 6 FNIII repeat exons. In addition to the transcriptional increase with high glucose, exon inclusion via alternative splicing was also changed resulting in production of higher molecular weight isoforms of TN-C. Mesangial cells grown in normal glucose secreted small isoforms with 1–2 variable repeats included whereas in high glucose large isoforms estimated to include 5 repeats were secreted. Unlike the smaller isoforms, the larger TN-C was not detected in the FN matrix. This change in TN-C isoforms may affect the regulation of FN matrix assembly and in this way may contribute to increased ECM accumulation under high glucose conditions.

Patterned hydrogel devices and methods for neural regeneration

Abstract: A matrix for neuron regeneration. The matrix can include a sheet having a first surface and a second surface opposite the first surface, the second surface having a plurality of integrally formed ridges. The sheet can have a spiral shape, such that the first surface of the sheet faces the second surface of the sheet. The sheet and the integrally formed ridges can comprise oligo(poly(ethylene glycol) fumarate).