Analysis of Wnt signaling β-catenin spatial dynamics in HEK293T cells

Abstract

BackgroundWnt/β-catenin signaling is involved in different stages of mammalian development and implicated in various cancers (e.g. colorectal cancer). Recent experimental and computational studies have revealed characteristics of the pathway, however a cell-specific spatial perspective is lacking. In this study, a novel 3D confocal quantitation protocol is developed to acquire spatial (two cellular compartments: nucleus and cytosol-membrane) and temporal quantitative data on target protein (e.g. β-catenin) concentrations in Human Epithelial Kidney cells (HEK293T) during perturbation (with either cycloheximide or Wnt3A). Computational models of the Wnt pathway are constructed and interrogated based on this data.ResultsA single compartment Wnt pathway model is compared with a simple β-catenin two compartment model to investigate Wnt3A signaling in HEK293T cells. When protein synthesis is inhibited, β-catenin decreases at the same rate in both cellular compartments, suggesting diffusional transport is fast compared to β-catenin degradation in the cytosol. With Wnt3A stimulation, the total amount of β-catenin rises throughout the cell, however the increase is initially (~first hour) faster in the nuclear compartment. While both models were able to reproduce the whole cell changes in β-catenin, only the compartment model reproduced the Wnt3A induced changes in β-catenin distribution and it was also the best fit for the data obtained when active transport was included alongside passive diffusion transport.ConclusionsThis integrated 3D quantitation imaging protocol and computational modeling approach allowed cell-specific compartment models of the signaling pathways to be constructed and analyzed. The Wnt models constructed in this study are the first for HEK293T and have suggested potential roles of inter-compartment transport to the dynamics of signaling.

Parameter-free methods distinguish Wnt pathway models and guide design of experiments

“…48), including conserved quantities. For example, Tan et al (22) measured average β-catenin in the cytoplasm (X) and the nucleus ðX n Þ after Wnt stimulation; we can investigate the relationship between these species. There are two models that include X and X n species: Schmitz et al and shuttle.…”

“…These three are allowed to vary and are used to fit the model to the Wnt/β-catenin time-course data recently published in ref. 22; see Materials and Methods for details. We chose the free parameters based on their point of influence on the pathway, targeting parameters with direct or near-direct influence on β-catenin dynamics (Fig.…”

“…From the data in ref. 22, we can test its compatibility with the model via a parameter-free method as described in ref. 24.…”

“…With the aim of discriminating between models, we present the five Wnt models under a unifying framework, with standardized notation to facilitate comparison. We fit parameters to recently published mammalian β-catenin signaling time-course data using Bayesian inference (22) and find that all of the studied models can describe the data well, demonstrating that additional data are required to compare models.…”

“…Experimental studies in Xenopus extracts have been performed to validate a model of Wnt signaling (5), with further pathway elucidation in refs. 19, 20; however, the parameters identified in these studies may differ markedly from those involved in mammalian Wnt signaling (21,22). With the aim of discriminating between models, we present the five Wnt models under a unifying framework, with standardized notation to facilitate comparison.…”