Biofunctional Nanodot Arrays in Living Cells Uncover Synergistic Co‐Condensation of Wnt Signalodroplets

Abstract

Qualitative and quantitative analysis of transient signaling platforms in the plasma membrane has remained a key experimental challenge. Here, biofunctional nanodot arrays (bNDAs) are developed to spatially control dimerization and clustering of cell surface receptors at the nanoscale. High-contrast bNDAs with spot diameters of 300 nm are obtained by capillary nanostamping of bovine serum albumin bioconjugates, which are subsequently biofunctionalized by reaction with tandem anti-green fluorescence protein (GFP) clamp fusions. Spatially controlled assembly of active Wnt signalosomes is achieved at the nanoscale in the plasma membrane of live cells by capturing the co-receptor Lrp6 into bNDAs via an extracellular GFP tag. Strikingly, co-recruitment is observed 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.

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

“…By contrast, co-clustering of Lrp6/Fzd8 into distinct signalosomes took place one order-of-magnitude slower timescale (~20 min), exhibiting distinct features of a nucleation-growth process. These characteristics are well in line with recent models suggesting receptor dimerization triggering the formation of canonical Wnt signalosomes by co-condensation with the scaffold proteins Axin and Dvl [35,37]. Strikingly, Ror2, the co-receptor for noncanonical Wnt signaling, which is not recognized by NGS, was found transiently trapped in NGS-induced canonical signalosomes.…”

“…Within these signalosomes, however, Lrp6 clearly showed mobility ( Fig. S5I-L , Movie S6 ), in line with condensate-like properties of these clusters, which we and others have recently proposed [37, 48, 49]. We further quantified Lrp6 confinement by pair correlation analysis [43, 47] ( Fig.…”

“…Previous studies had highlighted the indispensable roles of the cytosolic scaffold proteins in formation of the canonical Wnt signalosomes [29,[32][33][34]. Recent studies of Axin1 and Dvl2 confirmed these intrinsically disordered scaffold proteins co-condensate into protein droplets [35][36][37]. Moreover, puncta of Ror2, Fzd and Dvl2 in the plasma membrane were observed in Wnt5a-stimulated noncanonical signaling [27,38,39].…”

Interfacing Coacervates with Membranes: From Artificial Organelles and Hybrid Protocells to Intracellular Delivery

“…[56] For example, Wnt "signalodroplets" formed by LLPS that bind to the plasma membrane can amplify Wnt signaling. [57] Transmembrane proteins are not the only ones that can nucleate and localize condensates; nonspecific interactions between lipids and disordered proteins could also induce phase separation. Recently, Chatterjee et al showed that the presence of SUVs containing anionic DOPS and DOPG lipids could induce the formation of clusters of FUS low complexity (LC) domain and lipids at a 30-fold lower concentration than that needed for FUS LC phase separation in the absence of lipids, caused by FUS LC binding to SUVs and adopting a more ordered conformation (Figure 4e).…”

“…[ 56 ] For example, Wnt “signalodroplets” formed by LLPS that bind to the plasma membrane can amplify Wnt signaling. [ 57 ]…”