Quantitative live-cell imaging and computational modelling shed new light on endogenous wnt/ctnnb1 signaling dynamics

Abstract

WNT/CTNNB1 signaling regulates tissue development and homeostasis in all multi cellular 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 on cogenic 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 sub cellular compartments through three regulatory nodes: the destruction complex, nucleocyto plasmic shuttling and nuclear retention.