Characterization of the transcription factors SMAD1 SMAD5 and SMAD8 in the Bone Morphogenetic Protein (BMP) signaling pathway.

The bone morphogenic protein (BMP) pathway plays an essential role across an astonishingly large range of biological processes, from embryogenesis to cell homeostasis and apoptosis. One of the most interesting open questions about the BMP pathway is the signal reduction process. Information is converted from about 20 distinct extracellular BMP ligands, to only three intracellular transcription factors called SMADs. Here we hypothesize that a ligand specific information can be encoded by the combinatorics and dynamics of the three SMAD proteins. To allow a systematic view into this encoding we developed a quantitative microscopy based system that can determine the temporal dynamics of each SMAD individually. Specifically, we generated fluorescent reporter cell lines based on the NAMRU mouse mammary gland cells (NMuMG). These utilize a fluorescently labeled SMAD protein, allowing the analysis of the cytoplasm to nucleus translocation for each individual SMAD variant. A second reporter based on the BMP reporter element (BRE) allows measurement of the transcriptional activity of each cell. Using live fluorescence microscopy and advanced image analysis we tracked hundreds of individual cells and identified the main features that characterize the SMADs dynamics in response to a BMP ligand. Our study allows a dynamic view of the information flow within the BMP pathway and uncovers the encoding of information as well as the combinatorial role of the SMAD proteins.