Imaging transmembrane dynamics of biomolecules at live cell plasma membranes using quenchers in extracellular environment

It is a big challenge to measure position changes of biomolecules in the direction normal to the plasma membranes of living cells. We developed a one donor-multiple quenchers Fӧrster resonance energy transfer method by using non-fluorescent quenchers in the extracellular environment. It senses subnanometer position changes of a fluorophore-labeled biomolecule in the plasma membrane. The method was validated by monitoring flip-flops of individual lipid molecules incorporated in plasma membranes. We studies membrane perforation by a host defense peptide from the extracellular side and found that the pore-forming peptide is dynamic, switching among different insertion depths. The method is especially useful in studying interactions of membrane proteins with the inner surfaces of plasma membranes. Our method will find wide applications in systematic analysis of fundamental cellular processes at plasma membranes. ### Competing Interest Statement The authors have declared no competing interest.