Tan CW et al. (2012), Wnt signalling pathway parameters for mammalian...

XB-ART-44940

PLoS One 2012 Jan 01;72:e31882. doi: 10.1371/journal.pone.0031882.

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Wnt signalling pathway parameters for mammalian cells.

Tan CW , Gardiner BS , Hirokawa Y , Layton MJ , Smith DW , Burgess AW .

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Wnt/β-catenin signalling regulates cell fate, survival, proliferation and differentiation at many stages of mammalian development and pathology. Mutations of two key proteins in the pathway, APC and β-catenin, have been implicated in a range of cancers, including colorectal cancer. Activation of Wnt signalling has been associated with the stabilization and nuclear accumulation of β-catenin and consequential up-regulation of β-catenin/TCF gene transcription. In 2003, Lee et al. constructed a computational model of Wnt signalling supported by experimental data from analysis of time-dependent concentration of Wnt signalling proteins in Xenopus egg extracts. Subsequent studies have used the Xenopus quantitative data to infer Wnt pathway dynamics in other systems. As a basis for understanding Wnt signalling in mammalian cells, a confocal live cell imaging measurement technique is developed to measure the cell and nuclear volumes of MDCK, HEK293T cells and 3 human colorectal cancer cell lines and the concentrations of Wnt signalling proteins β-catenin, Axin, APC, GSK3β and E-cadherin. These parameters provide the basis for formulating Wnt signalling models for kidney/intestinal epithelial mammalian cells. There are significant differences in concentrations of key proteins between Xenopus extracts and mammalian whole cell lysates. Higher concentrations of Axin and lower concentrations of APC are present in mammalian cells. Axin concentrations are greater than APC in kidney epithelial cells, whereas in intestinal epithelial cells the APC concentration is higher than Axin. Computational simulations based on Lee's model, with this new data, suggest a need for a recalibration of the model.A quantitative understanding of Wnt signalling in mammalian cells, in particular human colorectal cancers requires a detailed understanding of the concentrations of key protein complexes over time. Simulations of Wnt signalling in mammalian cells can be initiated with the parameters measured in this report.

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Species referenced: Xenopus
Genes referenced: adm cdh1 ctnnb1 tbx2

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	Figure 1. 3D confocal imaging sample results (SW480APC).Trypsinised SW480APC cells stained with Calcein AM fluorescent dye (green), Hoechst 33342 nucleic acid stain (blue) and Vybrant DiI cell labelling solution (red), fluorescent marking the cytoplasm, nuclei and membrane respectively. Different overlayed fluorescent signal views of a sample of the acquired image stack are as show, namely (A) 2D sectional view; (B) orthogonal views of selected cells; (C) 3D volumetric view with blending effect.
	Figure 2. Population statistics of all cell types.Population statistics of all cells showing the (A) histograms of normalised volume (Calcein AM/Hoechst) and the (B) average normalised volume column chart. (AU: Arbitrary Units).
	Figure 3. Cell population sizing and selection.Population statistics of SW480APC, showing the (A) histograms of normalised volume (Calcein AM/Hoechst); (B) Scatter plot of fluorescent marked volumes (Calcein AM vs. Hoechst) showing a good linear relationship; (C) Scatter plot of fluorescent marked volumes (Calcein AM vs. Hoechst) showing population isolation and the cut-off volumes applied. Statistical results for other cell lines are as shown in the Figures S12, S13, S14, and S15.
	Figure 4. Cell volume measurement statistics for non-dividing cells.Fluorescent marked and calculated compartment volumes and distribution results showing (A) calibrated cell compartment volumes in pL, (B) distribution of compartment volume (% of total) and (C) fluorescent marked volumes in pL for non-dividing cells.
	Figure 5. Total protein per cell for each cell line.Error bar: Root Mean Squared Error (SEM).
	Figure 6. Key protein concentration distribution in mammalian cells.(A) Average concentration of key Wnt proteins and E-cadherin of the five mammalian cell lines. (B) Figure redrawn in linear scale, reiterating the significant difference in concentrations between the proteins measured and the effect of log scales on perception of concentration differences. *Note that the APC measured in Caco-2, SW480 and SW480 are ÎAPC. The amount of wild type APC is too low to be detected and the measurement here is for ÎAPC. In SW480APC, approximately 95% of the total APC is ÎAPC (unpublished data) (Data: Concentration in nM Â± SEM (n repeats), Error bars: SEM).
	Figure 7. Sub-cellular distribution of Î²-catenin in mammalian cells.(A) Compartment concentrations for Î²-catenin in HEK293T, Caco-2, MDCK, SW480 and SW480APC cells. Note: Percentage of whole cell Î²-catenin concentrations is in parentheses. (B) Protein recoveries (Sum of compartment proteins) from the fractionation experiment compared with the measured whole cell lysate.
	Figure 8. Concentrations of Axin and APC in mammalian whole cell lysate.The data is displayed in log scale. * Note that for SW480APC, as the level of wild-type (WT) APC is too low to be detected in this set of experiments, the measurements made in this study comprised that of mutant (MT) APC only. Error Bars: SEM.
	Figure 9. Key protein concentrations in different cell lines.Comparison of key protein concentrations (Î²-catenin, Axin, APC and GSK3Î²) between the Xenopus extract (Lee et al., 2003), two kidney epithelial mammalian cells (HEK293T and MDCK) and three intestinal epithelial mammalian cells (Caco-2, SW480 and SW480APC). * Î”APC measured in Caco-2, SW480 and SW480. For SW480APC, as the level of wild-type APC is too low to be detected in this set of experiment, the measurements made in this study comprised that of Î”APC only. The data is displayed in log scale.
	Figure 10. Computational Modelling Steps and Model Calibration Test Requirement.Typical computational modelling involves initial steady state analysis: Phase A, initial concentration to steady state protein redistribution in a closed system (no input and output from the system); Phase B, steady state protein redistribution in an open system and a subsequent transient analysis (Phase C, steady state to perturbation time-course). Phase A and B involves redistribution of proteins among the protein complexes while Phase C applies the redistributed concentrations to conduct transient investigations. Phase A and B are initial pre-requisites for a calibrated model whereby the total protein concentration for each protein should be maintained at steady state in an open system (inset).
	Figure 11. Steady State Analysis of protein complex for different cell lines.Initial to steady state protein redistribution among the interacting complexes of the Wnt signalling pathway for the different cell lines. (A) Xenopus (B) HEK293T (C) MDCK (D) Caco-2 (E) SW480 and (F) SW480APC. Note: Concentrations of protein and complexes involving Axin (in dotted box) are significantly low in all cell lines.
	Figure 12. Total protein concentrations of Î²-catenin and Axin.Initial and Steady State total concentrations for Xenopus egg extract (Lee et al., 2003) and whole cell lysate (WCL) from the mammalian cell lines. As expected, APC and GSK3Î² remained stable (results not shown) due to the lack of input and output fluxes in the Lee et al., 2003 model. Notes: For Î²-catenin, the ratio of total initial to steady state (Iâˆ¶SS) concentrations are given in the box below the bar for the respective cell line. Total protein concentration implies summation of concentrations of free and all protein complexes containing the specific protein.

References [+] :

Aberle, beta-catenin is a target for the ubiquitin-proteasome pathway. 1997, Pubmed