Format

Send to

Choose Destination
Matrix Biol. 2014 Feb;34:124-31. doi: 10.1016/j.matbio.2013.10.006. Epub 2013 Oct 19.

Heparin-dependent regulation of fibronectin matrix conformation.

Author information

1
Molecular Biology, Cell Biology & Biochemistry Program, Boston University, Boston, MA 02215, United States.
2
Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, United States.
3
Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, United States; Department of Biomedical Engineering, Boston University, Boston, MA 02215, United States; Department of Biological Sciences, University of Massachusetts Lowell, Lowell, MA 01854, United States. Electronic address: matthew_nugent@uml.edu.
4
Department of Biomedical Engineering, Boston University, Boston, MA 02215, United States. Electronic address: msmith@bu.edu.

Abstract

Extracellular matrix (ECM) conformation is regulated by a variety of stimuli in vivo, including mechanical forces and allosteric binding partners, and these conformational changes contribute to the regulation of cell behavior. Heparin and heparan sulfate, for example, have been shown to regulate the sequestration and presentation of numerous growth factors, including vascular endothelial growth factor, on the heparin 2 binding domain in fibronectin (Fn). However, mechanical force also alters Fn conformation, indicating that the growth factor binding region may be co-regulated by both heparin and mechanical force. Herein, we describe a simple antibody-based method for evaluating the conformation of the heparin 2 binding domain in Fn, and use it to determine the relative contributions of heparin and mechanical strain to the regulation of Fn conformation. We achieved specificity in quantifying conformational changes in this region of Fn by measuring the ratio of two fluorescent monoclonal antibodies, one that is insensitive to Fn conformational changes and a second whose binding is reduced or enhanced by non-equilibrium conformational changes. Importantly, this technique is shown to work on Fn adsorbed on surfaces, single Fn fibers, and Fn matrix fibers in cell culture. Using our dual antibody approach, we show that heparin and mechanical strain co-regulate Fn conformation in matrix fibrils, which is the first demonstration of heparin-dependent regulation of Fn in its physiologically-relevant fibrillar state. Furthermore, the dual antibody approach utilizes commercially available antibodies and simple immunohistochemistry, thus making it accessible to a wide range of scientists interested in Fn mechanobiology.

KEYWORDS:

Extracellular matrix; Fibronectin; Heparin

PMID:
24148804
PMCID:
PMC3992196
DOI:
10.1016/j.matbio.2013.10.006
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center