Direct Observation of Hop Diffusion of Lipid and Protein Molecules in the Plasma Membrane by High-Speed Single Fluorescent-Molecule Imaging

Abstract

Previously, using single-particle tracking at a temporal resolution of 0.025 ms, employing a 40-nmΦ colloidal gold probe, we have shown that virtually all of the lipid and protein molecules incorporated in the plasma membrane undergo hop diffusion. Based on this and many other observations, we proposed a model in which the entire plasma membrane is parcelled up into apposed domains due to the presence of the actin-based membrane skeleton (fence) and its associated transmembrane proteins (pickets), and membrane molecules undergo short-term confined diffusion within a domain (compartment), and long-term hop diffusion between the compartments. However, due to technological limitations, the observation of hop diffusion was only possible with a 40-nmΦ-colloidal gold probe, which might artifactually induce hop diffusion. To circumvent this problem, here, we developed a new high-speed, high-sensitivity CMOS camera system, which allowed us to track single fluorescently (0.5-nmΦ)-labeled molecules at a temporal resolution of 0.1 ms, the fastest single fluorescent-molecule imaging ever made. This camera system gave the position determination accuracy for single fluorescent molecules of ≈35 nm at a 0.1-ms time resolution. Virtually, all molecules of a phospholipid (DOPE) and a transmembrane protein, transferrin receptor, were found to undergo hop diffusion over the 110-nm compartments with median residency times of 9 ms and 33 ms, respectively, in the plasma membrane of a human epithelial T24 cell line. Meanwhile, in the actin-depleted, blebbed membrane, all of the DOPE and transferrin receptor molecules exhibited simple-Brownian diffusion. These results are in an excellent agreement with the previous high-speed gold-particle tracking data, and clearly indicate the necessity for the paradigm shift for the plasma membrane structure and dynamics, from the single continuous fluid model to the partitioned fluid model.