Biofunctional nanodot arrays in living cells uncover synergistic co-condensation of Wnt signalodroplets

Qualitative and quantitative analysis of transient signaling platforms in the plasma membrane has remained a key experimental challenge. Here, we have developed biofunctional nanodot arrays (bNDAs) to spatially control dimerization and clustering of cell surface receptors at nanoscale. High-contrast bNDAs with spot diameters of ~300 nm were obtained by capillary nanostamping of BSA bioconjugates, which were subsequently biofunctionalized by reaction with tandem anti-GFP clamp fusions. We achieved spatially controlled assembly of active Wnt signalosomes at the nanoscale in the plasma membrane of live cells by capturing the co-receptor Lrp6 into bNDAs via an extracellular GFP tag. Strikingly, we observed co-recruitment of co-receptor Frizzled-8 as well as the cytosolic scaffold proteins Axin-1 and Disheveled-2 into Lrp6 nanodots in the absence of ligand. Density variation and the high dynamics of effector proteins uncover highly cooperative liquid-liquid phase separation (LLPS)-driven assembly of Wnt signalodroplets at the plasma membrane, pinpointing the synergistic effects of LLPS for Wnt signaling amplification. These insights highlight the potential of bNDAs for systematically interrogating nanoscale signaling platforms and condensation at the plasma membrane of live cells.