Biophysical Society Thematic Meeting - June 28-July 1, 2015

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 o ) domains coexisting with liquid-disordered (L d ) phases can be visualized in fluorescence. The distinct L o 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 o 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 o raft domains is detected in sub-milliseconds, suggesting the membrane heterogeneities in the nanoscopic scale contribute to the regulation mechanisms of lipid rafts.

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