(775b) Measurement of the Dynamic Association/Dissociation of Membrane Biomolecules to Different Lipid Membrane Phases

Measurement of the dynamic association/dissociation of
membrane biomolecules to different lipid membrane phases

Ling Chao, Mark
Richards, and Susan Daniel

School of
Chemical and Biomolecular Engineering

Cornell
University, Ithaca, NY 14853

Separation of
distinct lipid membrane domains in cell membranes has been suggested to play
important roles in many cellular processes by providing various
microenvironments to cluster or to isolate membrane biomolecules. One class of
liquid-ordered membrane domain, enriched in sphingolipids and cholesterol, is called
a lipid raft. Lipid rafts have drawn a lot of attention due to their high
affinity for some signaling proteins. The residency of certain proteins inside
liquid-ordered phases has been show to have a significant impact on the protein
activity, compared to its activity when excluded from these phases.
Consequently, it is often postulated that the inclusion or exclusion of certain
biomolecules into or from lipid rafts might be a cellular mechanism for
regulating the molecule's activity and possibly its function, such as signaling.
This hypothesis is further bolstered by evidence that certain stimuli can cause
some biomolecules to change their residency in these domains, supporting
differential partitioning as a means to regulate protein activity levels. However,
most experimental determinations of biomolecule residency in rafts at various
conditions occur only at equilibrium conditions, but the cell is actually far
from equilibrium. In fact, determining critical lipid-protein interactions for
proper protein activity is extremely difficult to carry out in vivo.  Therefore, the objective of this work is to measure
the association/dissociation kinetics of membrane biomolecules with membrane
raft-like phases over shorter timescales in
vitro, information which to date has not been easy to obtain without
artifacts arising from the experimental procedures. To avoid many known artifacts,
we have developed a new approach for interrogating membrane residency and
measuring dynamics.  We employ a composite
membrane patterned with different membrane phases inside a microfluidic
channel. The key features of the heterogeneous supported bilayer that make this
technique possible are two-dimensional fluidity of the membrane constituents
and the ability to tune the bilayer chemistry and location of the domains. Fluorescently-labeled
biomolecules are loaded into the device and positions tracked in time using a
fluorescence microscopy to assay their partition kinetics into various
raft-like phases. We study the dynamics of three species with well-known domain
preferences: fluorescently-labeled lipids, glycolipids, and GPI-linked proteins.
We report their association and dissociation rate constants and compare their
ratio to their equilibrium partition coefficient.