A New Method for the Measurement of High Temporal Resolution Kinetics of Membrane Fusion to Supported Lipid Bilayers

A new method for the measurement of high temporal resolution kinetics of membrane fusion to supported lipid bilayers

Deirdre A. Costello and Susan Daniel

School of Chemical and Biomolecular Engineering

Cornell University, Ithaca, NY 14853

We developed a new in vitro method for studying biological membrane fusion that provides high temporal resolution through the rapid and coordinated initiation of individual fusion events. In this work, we focus on influenza virus fusion to synthetic supported lipid bilayers. Influenza is a membrane-enveloped virus, which necessitates the fusion of its membrane with the endosomal membrane of the cell in order to delivery its genetic material to the cytosol for infection. In nature, viral fusion to the endosomal membrane is initiated by a conformational change in the virus fusion protein, hemagglutinin, triggered by acidification of the endosome. Studying the kinetics of this process in vivo is difficult because fusion occurs inside an intracellular compartment after the virus is endocytosed by the cell. Therefore, we mimic the endosomal membrane chemistry in a supported bilayer coating the walls of a microfluidic device. We monitor hemifusion (the merging of the two outermost lipid leaflets) using fluorescence dequenching. Fusion between a fluorescently-labeled virus and the supported bilayer is initiated by a rapid decrease in pH by our method. Subsequent single particle fusion events are monitored with total internal reflection fluorescence microscopy.  Analysis of the stochastic events initiated by uncaging reveals a hemifusion rate constant at least an order of magnitude higher than previously reported. The increased sensitivity gained by this assay may facilitate comparison of fusion kinetics between different influenza strains to better characterize pandemic mutants and identify new potential targets (on the fusion protein) for antiviral drugs.