(Un)Confined Diffusion of CD59 in the Plasma Membrane Determined by High-Resolution Single Molecule Microscopy

Abstract

There has been emerging interest whether plasma membrane constituents are moving according to free Brownian motion or hop diffusion. In the latter model, lipids, lipid-anchored proteins, and transmembrane proteins would be transiently confined to periodic corrals in the cell membrane, which are structured by the underlying membrane skeleton. Because this model is based exclusively on results provided by one experimental strategy--high-resolution single particle tracking--we attempted in this study to confirm or amend it using a complementary technique. We developed a novel strategy that employs single molecule fluorescence microscopy to detect confinements to free diffusion of CD59--a GPI-anchored protein--in the plasma membrane of living T24 (ECV) cells. With this method, minimum invasive labeling via fluorescent Fab fragments was sufficient to measure the lateral motion of individual protein molecules on a millisecond timescale, yielding a positional accuracy down to 22 nm. Although no hop diffusion was directly observable, based on a full analytical description our results provide upper boundaries for confinement size and strength.

Membrane heterogeneity – from lipid domains to curvature effects

“…Those "pickets" are supposed to transiently confine proteins and lipids to nanometer sized domains (35). This idea has been challenged, by recent state of the art single-molecule fluorescence experiments (36), which do not find any confinement on length scales reported previously.…”

“…Gold is nontoxic and well-known surface chemistry allows them to be coupled to a wide range of molecules. The labeling ratio is, however, rather undefined and unspecific binding is an issue (36). Fluorescent quantum dots are considerably smaller than gold beads and possess good photostability (104,105) though blinking might be a problem for some applications.…”

“…However, the lack of high temporal resolution conceals transient processes. Those processes manifest themselves in anomalous diffusion of single molecules addressed by single-molecule or single-particle tracking experiments (35,36,(131)(132)(133). The achievable temporal resolution is typically a few ms for experiments in living cells (36,131).…”

“…Those processes manifest themselves in anomalous diffusion of single molecules addressed by single-molecule or single-particle tracking experiments (35,36,(131)(132)(133). The achievable temporal resolution is typically a few ms for experiments in living cells (36,131). Determination of kinetic parameters, PICS The recorded molecule positions are typically connected to trajectories (134)(135)(136) from which kinetic parameters are retrieved.…”

High-density mapping of single-molecule trajectories with photoactivated localization microscopy

“…By comparison, 10-100 cells are typically used to obtain statistically significant single-particle tracking information on individual proteins tagged with conventional labels because each cell yields less than 10 tracks on average [10][11][12] . We achieved similar statistics with sptPALM on a single cell (Fig.…”