Phosphatidylinositol 4,5-bisphosphate (PIP2) is a constitutional component of the cell membrane. It can directly or indirectly regulate vesicle trafficking, focal adhesion, cell migration and membrane-cytoskeleton interaction. It has been reported that sequestration of PIP2 in fibroblasts resulted in the decrease of the force needed to extract membrane tethers from the cells, a finding that was attributed to weakened membrane-cytoskeleton interaction, The commonly accepted mechanism of this phenomenon is that PIP2 inhibits the activity of actin-severing proteins such as cofilin, gelsolin, or profilin, and also activates vinculin, talin, α, β-catenin, α-actinin, and thus enhances the interaction between the cell membrane and cytoskeleton.The aim of our research is to clarify how PIP2 regulates endothelial cell function. Bovine aortic endothelial cells (BAECs) were treated with Pleckstrin homology domain of phospholipase C (PH-PLC) and Neomycin sulfate, which can sequester PIP2, and subsequently exposed to tether extraction with AFM. The results showed that after treatment with Neomycin sulfate, the tether force of BAEC slightly increased in comparison to control, while PH-PLC did not alter the tether force at all. These findings suggest that PIP2 functions differently in BAECs than in fibroblasts. These findings also suggest the following preliminary conclusions. (1) Sequestration of PIP2 with Neomycin might release actin binding proteins that may act as actin crosslinkers, thus strengthening the cytoskeleton in BAECs. (2) PH-PLC, may not be able to sequester PIP2, due to the latter's higher affinity to cytoskeleton binding proteins, thus not affecting the membrane-cytoskeleton interaction in BAEC.This work was supported by NIH-HL64388 (IL, G.F.) and the American Heart Association (M.S.)