OBSTRUCTED DIFFUSION AND VIRAL ADHESIVE INTERACTIONS IN PHASE SEPARATED LIPID BILAYERS

Tim Ratto (Ph.D. Biophysics U C Davis 2002)
Josetted Ricker (Ph.D. Biophysics U C Davis 2002)
Wan-chen Lin (graduate student, Biophysics Graduate Group)
Craig Blanchette (graduate student, Biophysics Graduate Group)
Natura Richardson (CPIMA SURE Program and Sacramento City College / U C Santa Cruz)

Recent Project Progress:
We have constructed a well-characterized model membrane system to better understand how nanometer-scale obstacles in cell membranes obstruct diffusion and for use in controlling the valency, mobility, and spacing of mobile or immobile ligands at the nanometer scale.  For these studies, we utilize supported bilayers composed of mixtures of 1,2-dilauroylphosphotidylcholine (DLPC) and 1,2 distearoylphosphotidylcholine (DSPC) or galactosyl ceramide (Gal-Cer), a saccharide lipid that is the major ligand for HIV virus trafficking in the sexual organs and mucosal system. Because these lipids are immiscible and phase separate at room temperature, a novel quenching technique allowed us to construct fluid DLPC bilayers containing small (~50 nm) disk-shaped gel-phase domains of DSPC or GalCer. Our experimental setup enabled us to analyze samples with atomic force microscopy and exactly characterize the size, shape, and number of gel-phase domains before measuring the obstacle-dependent diffusion coefficient (by fluorescence recovery after photobleaching) or binding of HIV gp120 (by total internal reflection fluorescence). Our diffusion data is used to validate and determine parameters in theories developed to explain diffusion coefficient measurements in cellular membranes.  Lateral obstructed diffusion was found to be dependent on obstacle area fraction, size, and geometry. We find that at solid-phase area fraction between ~35% and 70% (the percolation threshold), diffusion is anomalous at short times and becomes normal at longer times as predicted by theory and Monte Carlo simulations.  We have been able to control size and mobility in the GalCer domains and will discuss the binding of HIV gp120 to these domains. 

Project Publications:
“Obstructed Diffusion in Phase-Separated Supported Lipid Bilayers, A Combined AFM and FRAP Approach”, Biophysical Journal, Ratto, T. V. and Longo, M. L., 2002, 83: 3380-3392.
“Anomolous Subdiffusion in Heterogeneous Lipid Bilayers”, Ratto, T. V.  and Longo, M. L., Invited Research Article to Special Regular Issue of Langmuir on Biomolecular Interface, 2003, 19:1788 – 1793.
“Trehalose Maintains Phase Separation in an Air-Dried Binary Lipid Mixture” Biophysical Journal, Ricker, J., Tsvetkova, N., Wolkers, W., Leidy, C., Longo, M., and Crowe, J.H., 2003, 84: 3045-3051.

Description of Figures Below:
We developed a method to corral mobile lipids in bilayers simply by using phase separation of lipid mixtures.  We used a mixture of two lipids that are immiscible at room temperature.  One lipid (DLPC) has a phase transition below rt and the other lipid (DSPC) has a phase transition above rt.  A droplet of the 70 C vesicle solution was added to a freshly cleaved rt mica disk resulting in vesicle fusion to the mica and temperature quenching (Fig 1). This quenching process results in the formation of small lipid domains.    At low area fraction of DSPC, isolated DSPC disk shaped domains are present as seen by AFM (Fig. 2 – left image).  At an area fraction of ~55% the domains overlap and a change in geometry to extended disks is seen.  At higher area fractions (~70%) the percolation threshold (Fig. 8 - center image) is reached in agreement with the percolation threshold for extended disks and confined mobile lipid regions are formed (Fig. 2 – right image) above 70%.  Fluorescence recovery after photobleaching (Fig. 3) is used to characterize the diffusional behavior of this system.  The long-range diffusion coefficient decreases to zero (Fig. 4) at the percolation threshold of 70% confirming confinement of fluid bilayers.  The diffusional behavior is seen to become more time-dependent (anomolous diffusion) as the area fraction increases as predicted by Saxton (Fig. 5).