(322a) 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.