In the age of systems biology there is a need for more quantitative data for the “omics” approaches. In parallel, quantitative image analysis and data generation are advancing and allow for the development of better models of cellular processes such as regulation of signalling networks, protein movements and remodelling of cellular 3D architecture. Signalling networks such as the canonical Wnt-pathway regulate cellular differentiation which is controlled by TCF-dependent transcription. The activation of transcription depends on β-catenin translocation into the nucleus. We quantified β-catenin signals in different cellular compartments during the early differentiation phase of human neural progenitor cells VM 197 by semi-automatic quantitative analysis of confocal image stacks using Imaris™ software (Bitplane). To investigate the influence of Wnt signals on the β-catenin localization, the cells were treated with either activators or inhibitors of the Wnt signalling cascade: the glycogen synthase kinase-(GSK)-3β inhibitor SB216763, DKK1 or WIF-I (Wnt inhibitory factor-I) an inhibitor of free Wnt proteins. Wnt3a, a stimulator of the canonical Wnt-pathway, was used to investigate the upstream effects of this pathway. The results show that the nuclear β-catenin concentration increases pari passu with the induction of cell differentiation. Treatment with SB216763 and Wnt3a enhanced this effect, whereas DKK1 and WIF-I treatment suppressed it. The development of the neuronal phenotype was quantified in parallel in order to support the evidence that the differentiation is controlled by the Wntpathway. This precise quantification of fluorescence signals in 3D volumes correlated with neuronal phenotype development and, therefore, provides a means to investigate also other protein translocation processes between cellular compartments. Since the method is applicable to quantitatively describe different phenotypes the obtained data are well-suited to model cellular differentiation mechanisms correlated with spatio-temporal aspects of protein translocation.

17 – 21 May 2008