Ultrahigh-Speed Optical Imaging Unveils Single-Molecule Dynamics in Plasma Membrane of Live Cells
Yi-Hung Liao1*, Cheng-Hao Lin1, Chia-Lung Hsieh1
1Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan
* Presenter:Yi-Hung Liao, email:macgyver@gate.sinica.edu.tw
Plasma membrane is the barrier of living cells separating vital intracellular matrix and extracellular one and an important dynamic platform for functioning cellular machinery [1]. Macroscopic structure of cell membrane is widely accepted to be a variety of transmembrane and integral proteins embedded in a two-dimensional fluidic lipid bilayer. To date, nanoscale dynamics of these molecular constituents in the cell membrane are still unequivocally and incompletely answered. To gain insight of the nanoscopic events is challenging due to the difficulty of achieving simultaneous high submillisecond temporal resolution and nanometer spatial precision [2]. We attempt to resolve dynamics of single lipid and protein molecules in the plasma membrane of live cells with ultrahigh spatiotemporal resolution by direct optical microscope imaging and single molecule tracking. Small gold nanoparticle (as small as 20 nm in diameter) was used as the imaging probe. Monovalent labeling of the particle to the membrane molecule was achieved via monomeric avidin-like protein. Scattering signal from the gold nanoparticle was detected by imaging-based interferometry at 390,000 frames per second (corresponding to temporal resolution of 2.56 μs). Localization precision was estimated to be a few nanometers. Diffusion coefficient of single lipids probed by our monovalent gold probes is comparable to that reported using fluorescent dye at 2.5-3 μm²/s. Well characterized differences in lipid and protein diffusion in cell membranes treated with or without membrane skeleton destabilizer revealed by MSD analysis. In addition, trajectories of transferrin receptors showed an explore-and-retreat movement. Our data open a door to a better comprehension of the hypothesized existence of membrane nanodomains and nanoscopic confinements, and, even more, the interplay of molecular dynamics of lipids and proteins and the cellular skeletons.

[1] Simons K., and Ikonen E., Nature 387, 569-572 (1997).
[2] Simons K., and Sampaiohys J. L., Cold Spring Harb. Perspect. Biol. 3 (10), a004697 (2011).

Contact: Yi-Hung Liao (macgyver@gate.sinica.edu.tw)

Keywords: Single-Particle Tracking, Diffusion, Plasma membrane, Gold nanoparticles