β-catenin-Mediated Wnt Signal Transduction Proceeds Through an Endocytosis-Independent Mechanism

Abstract

The Wnt pathway is a key intercellular signaling cascade that regulates development, tissue homeostasis, and regeneration. However, gaps remain in our understanding of the molecular events that take place between ligand-receptor binding and target gene transcription. Here we used a novel tool for quantitative, real-time assessment of endogenous pathway activation, measured in single cells, to answer an unresolved question in the field – whether receptor endocytosis is required for Wnt signal transduction. We combined knockdown or knockout of essential components of Clathrin-mediated endocytosis with quantitative assessment of Wnt signal transduction in mouse embryonic stem cells (mESCs). Disruption of Clathrin-mediated endocytosis did not affect accumulation and nuclear translocation of β-catenin, as measured by single-cell live imaging of endogenous β-catenin, and subsequent target gene transcription. Disruption of another receptor endocytosis pathway, Caveolin-mediated endocytosis, did not affect Wnt pathway activation either. These results, confirmed in multiple cell lines, suggest that endocytosis is not a general requirement for Wnt signal transduction. We show that off-target effects of a drug used to inhibit endocytosis may be one source of the discrepancy among reports on the role of endocytosis in Wnt signaling.

Wnt signalling and endocytosis: Mechanisms, controversies and implications for stress responses

“…This study, while supporting a role for endocytosis in Wnt signal transduction, argues against the involvement of clathrin, AP2 and other clathrin‐associated molecules. Further complicating the issue, a recent study by Rim, Nusse and colleagues reported that neither clathrin‐dependent nor caveolin‐mediated endocytosis was required for Wnt signal transduction in multiple mammalian cell types (Rim et al., 2020). Using a mouse embryonic stem cell line in which the endogenous β‐catenin was tagged with a Venus fluorescent epitope, they showed that nuclear translocation of β‐catenin could proceed in the presence of knockdown or knockout of the clathrin heavy chain gene (Rim et al., 2020).…”

“…Further complicating the issue, a recent study by Rim, Nusse and colleagues reported that neither clathrin‐dependent nor caveolin‐mediated endocytosis was required for Wnt signal transduction in multiple mammalian cell types (Rim et al., 2020). Using a mouse embryonic stem cell line in which the endogenous β‐catenin was tagged with a Venus fluorescent epitope, they showed that nuclear translocation of β‐catenin could proceed in the presence of knockdown or knockout of the clathrin heavy chain gene (Rim et al., 2020). Moreover, the cell membrane abundance of several FZDs and LRP5/6, assessed by labelling of a FZD‐specific antibody and surface biotinylation, respectively, was unchanged by knockdown of clathrin heavy chain (Rim et al., 2020).…”

“…Using a mouse embryonic stem cell line in which the endogenous β‐catenin was tagged with a Venus fluorescent epitope, they showed that nuclear translocation of β‐catenin could proceed in the presence of knockdown or knockout of the clathrin heavy chain gene (Rim et al., 2020). Moreover, the cell membrane abundance of several FZDs and LRP5/6, assessed by labelling of a FZD‐specific antibody and surface biotinylation, respectively, was unchanged by knockdown of clathrin heavy chain (Rim et al., 2020).…”

“…First, many studies of Wnt signal transduction employ overexpression of either wild‐type or mutant Wnt pathway components, which could engage mechanisms that do not operate under physiological conditions. Second, reagents that claim to modulate endocytosis, such as monodansylcadaverine, may inhibit Wnt signalling independent of endocytosis (Rim et al., 2020). However, the new study does acknowledge that Wnt signal transduction could engage endocytosis in specific cellular and physiological contexts, especially when noncanonical Wnt receptors are involved, such as the epidermal growth factor receptor (Grainger et al., 2019).…”

Quantitative live-cell imaging and computational modelling yield novel insight into endogenous WNT/CTNNB1 signaling dynamics

“…This can severely affect activation, localization and complex formation (Gibson et al, 2013; Mahen et al, 2014). Although stabilization of CTNNB1 by WNT signaling has been extensively studied, very few studies have focused on the spatiotemporal dynamics of this process – especially at the endogenous level (Chhabra et al, 2019; Massey et al, 2019; Rim et al, 2020).…”