Transport of Fibroblast Growth Factor 2 in the Pericellular Matrix Is Controlled by the Spatial Distribution of Its Binding Sites in Heparan Sulfate

Vivien Octeau,Ian A Prior,Alison Beckett,Rachel N Bearon,Arthur D Lander,David G Fernig,Laurence Duchesne,Brahim Lounis

doi:10.1371/journal.pbio.1001361

Abstract

Author SummaryThe development, homeostasis, and repair of animal tissues requires communication between cells mediated by effector proteins, which are released from source cells and must move through the surrounding extracellular matrix to reach their receptors on target cells. A major component of the extracellular matrix is the polysaccharide heparan sulfate (HS); it binds the majority of these effectors and has the crucial function of regulating their transport. The mechanism underlying this function, however, is still unknown. To understand how HS regulates the transport of effectors, in this study we labelled molecules of the effector protein fibroblast growth factor 2 (FGF2) each with a gold nanoparticle, which we could visualise and quantify by electron microscopy and by a new approach called photothermal heterodyne imaging. By imaging the gold nanoparticles, we found that the binding sites for FGF2 on HS are distributed heterogeneously in the extracellular matrix that surrounds cells in culture. Single molecule tracking indicated that these binding sites are organised into local networks that confine the FGF2 and into paths that allow its translocation over long distances (up to several micrometers). Thus, the spatial distribution of the binding sites in HS and their physicochemical properties of binding are major factors controlling the transport of effectors in extracellular matrices.

Highlights

The notion of gradients of morphogens and of epithelialmesenchymal signal relays is common currency in developmental biology [1,2,3,4]
In the absence of exogenous heparin-derived dodecasaccharide (DP12), we show that virtually all fibroblast growth factor 2 (FGF2) bound to the pericellular matrix is engaged with heparan sulfate (HS), rather than the fibroblast growth factors (FGF) receptor (FGFR)
The Nterminus of FGF2 is an appropriate location for conjugation of a probe, because it is opposite the binding site for FGFR and the canonical heparin binding site and there are natural N-terminal extensions of FGF2 that do not affect its ability to bind heparin and activate FGFRs [30,31,32]

Summary

Introduction

The notion of gradients of morphogens and of epithelialmesenchymal signal relays is common currency in developmental biology [1,2,3,4]. Organism homeostasis often depends on similar transport of effector proteins, such as growth factors, cytokines, and chemokines from source to target cell, for example, in wound repair and in the regulation of immune responses [5] Such transport occurs in the extracellular matrix that lies between cells, including the pericellular matrix adjacent to the plasma membrane, where the heparan sulfate (HS) chains of proteoglycans (PGs) are the dominant molecular species [6]. This dominance is due to their size (,40 nm to 160 nm long), amount, and unlike the other extracellular glycans, their large array of protein partners (over 400), which they bind with varying degrees of selectivity [7,8]. A particular feature is the long, unbranched glycosaminoglycan chain, in which tracts of variably sulfated saccharides, responsible for the interaction with proteins, Author Summary

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