Long-term single molecule localization microscopy uncovers dynamic co-assembly of Lrp6 and Ror2 into Wnt-signalosomes

Abstract

The conserved Wnt signaling has been classified as two categories of canonical and noncanonical Wnt signaling. With a high promiscuity of Wnt signaling, how receptors from the two distinct pathways re-arrange in multi-protein signalosomes remains elusive. We here developed single-molecule tracking and localization microscopy based on labeling with reversibly binding nanobodies (rbTALM) for imaging receptor dynamics in the plasma membrane for extended time periods. To this end, we engineered nanobody-tag pairs with fine-tuned binding stabilities ensuring single-molecule tracking with high fidelity, yet continuous exchange of photobleached labels. Multicolor rbTALM imaging enabled simultaneous tracking and super-resolution imaging of three different Wnt co-receptors in the same cell for more than one hour at video rate. Time-lapse correlation analyses uncovered cooperative association of canonical and noncanonical Wnt co-receptors into a common, hybrid Wnt signalosome, demonstrating the exciting possibilities of rbTALM imaging for exploring nanoscale dynamics across millisecond to hour timescales.

Long-term single-molecule tracking in living cells using weak-affinity protein labeling

“…several tens of minutes, and which could previously only be accessed by measuring multiple cells sequentially [13,28] or limited to extracellular target sites. [23,43] To demonstrate the potential of the xHTL/HT7 labeling pair, we conducted SPT experiments with EGFR-HT7 in live cells. We chose EGFR as it is a membrane receptor that has been extensively studied with various SPT methods.…”