Abstract

Heparan sulfate (HS) chains of proteoglycans (PG) are major components of pericellular and extracellular matrices. They regulate transport, gradient formation and effector functions of >250 proteins central to animal cell communication: contrasting mechanisms proposed for this range from cyclic protein-HS association/dissociation to protein sequestration by HS that requires enzymatic liberation. Previous measurements averaging across large numbers of protein molecules interacting with HS do not resolve these explanations. To address this, we stoichiometrically labelled single molecules of archetypal HS-binding morphogen, fibroblast growth factor 2 (FGF2) with gold nanoparticles and used photothermal heterodyne imaging to track individual molecules in the pericellular matrix. We show individual FGF2 molecules undergo mainly local motion in the matrix (~65 nm radius) from which they can escape by diffusion (slow, fast or directed). Similar molecular motion persists when membrane PG movement is impeded by cell fixation: this shows FGF2 can slide along >10 successive HS chains driven by thermal energy and the observed heterogeneously located binding sites on HS chains. We conclude morphogen transport in pericellular matrix involves multiple mechanisms: sliding along HS chains, local transfer between binding sites on neighbouring chains and diffusion of the PG core protein within the membrane, which can be cytoskeleton driven