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.