Multiple signaling pathways, including Wnt signaling, participate in animal development, stem cell biology, and human cancer. Although many components of the Wnt pathway have been identified, unresolved questions remain as to the mechanism by which Wnt binding to its receptors Frizzled and Low-density lipoprotein receptor-related protein 6 (LRP6) triggers downstream signaling events. With live imaging of vertebrate cells, we show that Wnt treatment quickly induces plasma membrane-associated LRP6 aggregates. LRP6 aggregates are phosphorylated and can be detergent-solubilized as ribosome-sized multiprotein complexes. Phospho-LRP6 aggregates contain Wnt-pathway components but no common vesicular traffic markers except caveolin. The scaffold protein Dishevelled (Dvl) is required for LRP6 phosphorylation and aggregation. We propose that Wnts induce coclustering of receptors and Dvl in LRP6-signalosomes, which in turn triggers LRP6 phosphorylation to promote Axin recruitment and beta-catenin stabilization.

In biological microscopy, the ever expanding range of applications requires quantitative approaches that analyze several distinct £uorescent molecules at the same time in the same sample. However, the spectral properties of the £uorescent proteins and dyes presently available set an upper limit to the number of molecules that can be detected simultaneously with common microscopy methods. Spectral imaging and linear unmixing extends the possibilities to discriminate distinct £uoro-phores with highly overlapping emission spectra and thus the possibilities of multicolor imaging. This method also o¡ers advantages for fast multicolor time-lapse microscopy and £uores-cence resonance energy transfer measurements in living samples. Here we discuss recent progress on the technical implementation of the method, its limitations and applications to the imaging of biological samples. ß