Calcium signals at cytoneme contacts amplify Wnt/β-catenin signalling

Abstract

Wnt signalling controls morphogenesis, patterning and homeostasis across tissues. Because Wnt ligands are lipid-modified, their movement between cells cannot rely on simple diffusion and instead requires specialised transport mechanisms. Signalling filopodia, also known as cytonemes, have emerged as a key mediator of contact-dependent morphogen delivery. The mechanisms that stabilise signalling interfaces between Wnt ligand-bearing and receptor-bearing membranes at the cytoneme contact sites, a prerequisite for efficient Wnt ligand-receptor complex assembly and downstream pathway activation, remain poorly understood. Here, we show that HeLa cell cytonemes carry canonical Wnt components and form contact sites marked by focal Ca2+ transients. At these contacts, local Wnt and Ca2+ signals act in a positive feedback loop to promote longer-lasting interactions between neighbouring cells. Mechanistically, we find that E-cadherin/Cadherin 1 (CDH1) co-accumulates at these cytoneme contact sites in a Ca2+ - and Wnt-dependent manner, suggesting that contact stabilisation can be mediated by local adhesion. Consistent with this, presentation of Wnt ligands and CDH1 at cytoneme tips, together with Ca2+ availability, enhances Wnt signalosome formation at contact sites and, consequently, nuclear beta-catenin accumulation in the target cells. Our data support a model in which local Ca2+ transients stabilise cytoneme contacts and facilitate efficient Wnt ligand transfer to the receiving membrane, thereby enhancing canonical Wnt/beta-catenin signalling. We propose that focal Ca2+ signalling can serve as a contact-authentication step for morphogen signalling, converting transient cytoneme encounters into effective activators of the signal transduction cascade.