Heparan sulfate determines the modes of diffusion of fibroblast growth factor 2 within the pericellular matrix

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</p

Supplemental Movie 1: Time-lapse imaging in hypoxia of a single HeLa cell ectopically expressing EGFP-HIF-2α. from Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

Cellular adaptation to hypoxia occurs via a complex program of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity

Supplemental movie 5: Half-nucleus bleaching of a cell expressing EGFP-HIF-2α from Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

Cellular adaptation to hypoxia occurs via a complex program of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity

Supplental Movie 2:Fast imaging of a HeLa cell ectopically expressing EGFP-HIF-2α. from Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

Cellular adaptation to hypoxia occurs via a complex program of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity

Supplemental movie 7 from Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

Cellular adaptation to hypoxia occurs via a complex program of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity

Supplental Movie 3: Trajectory of individual HIF-2α speckle in single cell (example 1). from Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

Cellular adaptation to hypoxia occurs via a complex program of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity