Lin, S.; Ball, D. A.; Fazel, M.; Karpova, T. S.; Ho, M.

Glypican-3 (GPC3) is a heparan sulfate proteoglycan that is highly expressed in hepatocellular carcinoma and promotes tumor progression through Wnt3a/{beta}-catenin signaling. However, how the nanoscale organization of GPC3 at the cell surface controls signaling remains unclear. Here, we combined nano-resolution MINFLUX imaging, single-molecule tracking, and functional assays to define the spatial architecture and dynamics of GPC3 on hepatoma cells. We found that GPC3 exists as both single molecules and nanoscale clusters and switches between confined and free diffusions on the plasma membrane. Heparan sulfate (HS) chains create nanoscale corrals that limit GPC3 movement, whereas removal of HS increases diffusive heterogeneity and disrupts confinement. Wnt3a stimulation induces the formation of higher-order GPC3 assemblies and enhances {beta}-catenin signaling, while loss of HS markedly reduces this response. MINFLUX DNA-PAINT further revealed that HS chains orchestrate the spatial distribution of Wnt3a and promote its association with the Wnt receptor, Frizzled-1, an essential step for pathway activation. Collectively, these findings reveal that HS controls the nanoscale organization and dynamics of GPC3 to promote Wnt receptor assembly and efficient {beta}-catenin signaling in hepatoma cells.