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SLLISWD sequence in the 10FNIII domain initiates fibronectin fibrillogenesis.

ABSTRACT: Fibronectin (FN) assembly into extracellular matrix is tightly regulated and essential to embryogenesis and wound healing. FN fibrillogenesis is initiated by cytoskeleton-derived tensional forces transmitted across transmembrane integrins onto RGD binding sequences within the tenth FN type III (10FNIII) domains. These forces unfold 10FNIII to expose cryptic FN assembly sites; however, a specific sequence has not been identified in 10FNIII. Our past steered molecular dynamics simulations modeling 10FNIII unfolding by force at its RGD loop predicted a mechanical intermediate with a solvent-exposed N terminus spanning the A and B ?-strands. Here, we experimentally confirm that the predicted 23-residue cryptic peptide 1 (CP1) initiates FN multimerization, which is mediated by interactions with 10FNIII that expose hydrophobic surfaces that support 8-anilino-1-napthalenesulfonic acid binding. Localization of multimerization activity to the C terminus led to the discovery of a minimal 7-amino acid "multimerization sequence" (SLLISWD), which induces polymerization of FN and the clotting protein fibrinogen in addition to enhancing FN fibrillogenesis in fibroblasts. A point mutation at Trp-6 that reduces exposure of hydrophobic sites for 8-anilino-1-napthalenesulfonic acid binding and ?-structure formation inhibits FN multimerization and prevents physiological cell-based FN assembly in culture. We propose a model for cell-mediated fibrillogenesis whereby cell traction force initiates a cascade of intermolecular exchange starting with the unfolding of 10FNIII to expose the multimerization sequence, which interacts with strand B of another 10FNIII domain via a Trp-mediated ?-strand exchange to stabilize a partially unfolded intermediate that propagates FN self-assembly.

SUBMITTER: Gee EP

PROVIDER: S-EPMC3774401 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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SLLISWD sequence in the 10FNIII domain initiates fibronectin fibrillogenesis.

Gee Elaine P S EPS Yüksel Deniz D Stultz Collin M CM Ingber Donald E DE

The Journal of biological chemistry 20130605 29

Fibronectin (FN) assembly into extracellular matrix is tightly regulated and essential to embryogenesis and wound healing. FN fibrillogenesis is initiated by cytoskeleton-derived tensional forces transmitted across transmembrane integrins onto RGD binding sequences within the tenth FN type III (10FNIII) domains. These forces unfold 10FNIII to expose cryptic FN assembly sites; however, a specific sequence has not been identified in 10FNIII. Our past steered molecular dynamics simulations modeling ...[more]

PMID: 23740248

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Project description:We previously developed a surface-assisted assay to image early steps of cell-induced plasma fibronectin (FN) fibrillogenesis by timelapse atomic force microscopy (AFM). Unexpectedly, complementary attempts to visualize FN fibrillogenesis using fluorescently labeled FN (Alexa Fluor 488 or 568) and live-cell light microscopy initially failed consistently. Further analysis revealed that fibrillar remodeling was inhibited efficiently in the focal area illuminated during fluorescence imaging, but progressed normally elsewhere on the substrate, suggesting photo sensitivity of the FN fibrillogenesis process. In agreement, active cell-driven fibrillar extension of FN could be stopped by transient illumination with visible light during AFM timelapse scanning. Phototoxic effects on the cells could be ruled out, because pre-illuminating the FN layer before cell seeding also blocked subsequent fibrillar formation. Varying the illumination wavelength range between 400 and 640 nm revealed strong inhibition across the visible spectrum up to 560 nm, and a decreasing inhibitory effect at longer wavelengths. The photo effect also affected unlabeled FN, but was enhanced by fluorophore labeling of FN. The inhibitory effect could be reduced when reactive oxygen species (ROS) were removed for the cell imaging medium. Based on these findings, FN fibrillogenesis could be imaged successfully using a labeling dye with a long excitation wavelength (Alexa Fluor 633, excitation at 632 nm) and ROS scavengers, such as oxyrase, in the imaging medium. Fibrillar remodeling of exposed cell-free FN layers by AFM scanning required higher scan forces compared to non-exposed FN, consisting with mechanical stiffing of the FN layer after illumination. In agreement with changes in FN mechanics, cells spreading on pre-exposed FN showed reduced migration speeds, altered focal adhesion arrangement, and changes in mechanosensitive signaling pathways, including reduced FAK (Y397) and paxillin (Y118) phosphorylation. Pre-exposure of FN to visible light prior to cell seeding thus provides a useful tool to delineate mechanosensitive signaling pathway related to FN fibrillogenesis. When using FN-coated cell adhesion substrates, care should be taken when comparing experimental results obtained on non-exposed FN layers in cell culture incubators, or during live-cell fluorescence imaging, as FN fibrillogenesis and mechanosensitive cellular signaling pathways may be affected differently.

Dataset Information

SLLISWD sequence in the 10FNIII domain initiates fibronectin fibrillogenesis.

Publications

SLLISWD sequence in the 10FNIII domain initiates fibronectin fibrillogenesis.

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