Hour-scale single-molecule imaging reveals dynamic assembly of the Wnt co-receptors LRP6 and ROR2 into common signalosomes

Wnt ligands stimulate β-catenin-dependent (canonical) or β-catenin-independent (noncanonical) signaling, depending on which co-receptors are recruited to the Wnt receptor FZD. Both pathways are initiated by receptor oligomerization into signalosomes and involve a largely overlapping set of downstream effectors. To resolve the assembly of Wnt signalosomes with high spatiotemporal resolution for extended times, we developed single-molecule tracking and localization microscopy based on labeling with reversibly binding nanobodies (rbTALM). We engineered nanobody-tag pairs with finely tuned binding affinities to ensure single-molecule tracking with high fidelity while also permitting continuous exchange of photobleached labels. Multicolor rbTALM imaging enabled simultaneous tracking and super-resolution imaging of three different tagged Wnt co-receptors in the same cell for more than 1 hour at video rate. Time-lapse correlation analyses uncovered cooperative association of canonical (LRP6) and noncanonical (ROR2) Wnt co-receptors with FZD8 into a common, hybrid Wnt signalosome. These findings demonstrate the potential for rbTALM imaging for exploring nanoscale dynamics across millisecond to hour timescales and for deciphering the molecular dynamics that underlie signaling complex formation.