PROJECT SUMMARY During pregnancy, the proper development of the placenta is essential to maintain the health of the fetus and mother; however, we still do not have a clear understanding of how certain components of the placenta normally form. We are interested in how the synctitiotrophoblast barrier is formed through cellular fusion, and to understand this process, we will focus on the proteins presented on the outside of the barrier called cell surface proteins (CSPs). CSPs are regulators of cell-cell interactions and help cells respond to their environment, making them biologically important and relevant drug targets. However, CSPs are challenging to study, because they are in low abundance and are covered with sugar molecules called glycans. These glycans are highly variable structures that cannot be predicted, requiring experimental evidence to ascertain their composition. One of the most important functions of these glycans is to interact with glycan-binding proteins (GBPs). GBPs bind to glycans on the cell surface, resulting in downstream changes to cell behavior. GBPs, like galectin-3, have been shown to be necessary for normal fetal development and placental cell fusion, but discovering their mechanism of action is challenging, as the weak and dynamic interactions between glycans and GBPs are not easy to capture. To overcome these limitations, we have developed two new techniques that allow us to label and identify CSPs and galectin-3 interactors in live cells. We will apply these methods to a model of placental cell fusion to understand the factors that lead to successful barrier formation. Once critical CSPs and galectin-3 interactors are uncovered, we will perform genetic and biochemical experiments to determine their functions in detail. We will map the signaling pathways within the cell that allow for fusion, as well as the composition of glycans that allow for GBP interactions.