Quantitative live-cell imaging and computational modeling shed new light on endogenous WNT/CTNNB1 signaling dynamics.

Saskia Ma De Man,Mark A Hink,Gooitzen Zwanenburg,Renée Van Amerongen,Tanne Van Der Wal

doi:10.7554/elife.66440

Saskia Ma De Man, Mark A Hink + Show 3 more

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https://doi.org/10.7554/elife.66440

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Abstract

WNT/CTNNB1 signaling regulates tissue development and homeostasis in all multicellular animals, but the underlying molecular mechanism remains incompletely understood. Specifically, quantitative insight into endogenous protein behavior is missing. Here, we combine CRISPR/Cas9-mediated genome editing and quantitative live-cell microscopy to measure the dynamics, diffusion characteristics and absolute concentrations of fluorescently tagged, endogenous CTNNB1 in human cells under both physiological and oncogenic conditions. State-of-the-art imaging reveals that a substantial fraction of CTNNB1 resides in slow-diffusing cytoplasmic complexes, irrespective of the activation status of the pathway. This cytoplasmic CTNNB1 complex undergoes a major reduction in size when WNT/CTNNB1 is (hyper)activated. Based on our biophysical measurements, we build a computational model of WNT/CTNNB1 signaling. Our integrated experimental and computational approach reveals that WNT pathway activation regulates the dynamic distribution of free and complexed CTNNB1 across different subcellular compartments through three regulatory nodes: the destruction complex, nucleocytoplasmic shuttling, and nuclear retention.

Highlights

WNT signaling is one of the most ancient pattern-forming cell signaling cascades
Using Fluorescence Correlation Spectroscopy (FCS) and Number and Brightness (N&B) analysis we measure the mobility and concentration of CTNNB1, providing detailed information on CTNNB1 containing complexes in the cytoplasm and nucleus. We use these biophysical parameters to build a computational model of WNT/CTNNB1 signaling that predicts the levels and subcellular distribution of CTNNB1 across its cytoplasmic and nuclear pools. Using this integrated experimental and computational approach, we find that WNT regulates the dynamic distribution of CTNNB1 across different functional pools by modulating three regulatory nodes: cytoplasmic destruction, nucleocytoplasmic shuttling and nuclear retention
When WNT is present in the system, we describe the inactivation of the destruction complex (‘DC*’) by DVL through the parameter w

Summary

Introduction

WNT signaling is one of the most ancient pattern-forming cell signaling cascades. It drives many biological processes from the onset of embryogenesis until adulthood in all multicellular animals (reviewed in van Amerongen and Nusse, 2009; Holstein, 2012; Loh et al., 2016). In the absence of WNT signaling, rapid turnover by the so-called destruction complex maintains low levels of CTNNB1 This cytoplasmic complex consists of the scaffold proteins Adenomatous Polyposis Coli Protein (APC) and Axis Inhibition Proteins 1 and 2 (AXIN), which bind CTNNB1, and the serine/threonine kinases Casein kinase I isoform alpha (CSNK1A1) and Glycogen Synthase Kinase-3 alpha and beta (GSK3), which subsequently phosphorylate residues S45, T41, S37 and S33 (Amit et al, 2002; Liu et al, 2002). This allows newly synthesized CTNNB1 to accumulate and translocate to the nucleus, where CTNNB1 binds to TCF/LEF transcription factors (TCF7, TCF7L1, TCF7L2 and LEF1) to regulate target gene transcription as part of a larger transcriptional complex (Behrens et al, 1996; Fiedler et al., 2015; Molenaar et al, 1996; van Tienen et al, 2017)

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