Quantifying BMP2 Dynamics during Stem Cell Differentiation using, Real Time Imaging, Combined Confocal AFM and Systems Biology Approaches

Abstract

Bone morphogenetic proteins (BMP2) are key regulators during mesenchymal stem (MSC) cell differentiation; however how these growth factors achieve lineage specification is not well understood. BMP-2, a frequently studied BMP, is known to induce adipogenesis, chondrogenesis and osteogenesis in MSCs, however the exact dynamics leading to the determination of the fate of the stem cell is not well understood. We hypothesize those regions on the plasma membrane, where BMP-2 binds direct signaling pathway and therefore regulate stem cell fate.In order to determine the dynamics of BMP2 and binding sites on the plasma membrane we covalently link Quantum Dots (QDs) to BMP-2. Creating stable fluorescent BMP2 allowed for the first time measurements of BMP2 dynamics on the cell surface in real time. QD’s, semi-conducting nanocrystals, superior to conventional organic dyes, are highly efficient due to high extinction coefficient and stability (minimizing photo bleaching), which are 20 times brighter and 100 times more stable than traditional fluorescent reporters. Additionally results were verified using combined AFM and Confocal imaging. Using mutated RFP tagged BMP type - I receptors, or gfp tagged caveolin-1 or AP-2 constructs we were able to identify different regions important for osteogenesis, chondrogenesis and adipogenesis. Further using these data combined with a systems biology approach created a signaling model for BMP2 during stem cell differentiation.Our data indicate BMP2 interacts with receptors localized in caveolae and clathrin coated pits and these domains are key regions for signaling. Dependent on BMP receptor localization MSCs are directed toward specific stem cell fate.