Biophysical Society Thematic Meeting

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Poster Abstracts

48-POS

Board 48

Direct Visualization of Nanoscopic Diffusion Path of Single Lipid Molecules in Raft-

Containing Bilayer Membranes with Microsecond Temporal Resolution

Hsiao-Mei Wu

, Ying-Hsiu Lin, Tzu-Chi Yan, Chia-Lung Hsieh.

Academia Sinica, Taipei, Taiwan.

Lipid rafts are lipid domains that float dynamically on cell plasma membranes. They are believed

to play an important role in cell signaling processes due to their capability of recruiting receptor

molecules into domains through interaction between lipids and proteins. Experimental evidence

of how such interaction takes place at single-molecule level is limited because it is challenging to

obtain sufficient spatiotemporal resolution for observing rapid and nanoscopic motion of

individual molecules in the heterogeneous membranes.

Here, we employ an advanced high-speed optical microscope along with the single-particle

tracking method to directly observe the partition and diffusion of single molecules in raft-

containing reconstituted bilayer membranes. The bilayer membranes consisted of micron-sized

liquid-ordered (L

) domains coexisting with liquid-disordered (L

) phases can be visualized in

fluorescence. The distinct L

domains provide a platform for studying lipid rafts as they share

features with raft domains in cell membranes. Using interferometric detection of scattering signal

(iSCAT) technique, we image and track individual small gold particles (20 nm in diameter)

attached to the head groups of the probe lipid molecules. The iSCAT detection provides optimal

spatial localization precision down to a few nanometers at very high speed (50 kHz), which

allows us to continuously follow the motion of single lipid molecules with microsecond temporal

resolution. The precise diffusion trajectories of the probe lipids were overlaid on the

corresponding fluorescence image indicating the location of the L

domains. The high

spatiotemporal resolution reveals lipid diffusion in and out of membrane domains with

unprecedented clarity. By further analyzing the trajectories, subdiffusion of lipids in L

raft

domains is detected in sub-milliseconds, suggesting the membrane heterogeneities in the

nanoscopic scale contribute to the regulation mechanisms of lipid rafts.