Direct observation of brownian motion of lipids in a membrane.

Abstract

Nanovid microscopy, which uses 30-to 40-nm colloidal gold probes combined with video-enhanced contrast, can be used to examine random and directed movements of individual molecules in the plasma membrane of living cells. To validate the technique in a model system, the movements of lipid molecules were followed in a supported, planar bilayer containing fluorescein-conjugated phosphatidylethanolamine (Fl-PtdEtn) labeled with 30-nm gold anti-fluorescein (anti-Fl). Multivalent gold probes were prepared by conjugating only anti-Fl to the gold. Paucivalent probes were prepared by mixing an irrelevant antibody with the anti-Fl prior to conjugation. The membrane-bound gold particles moved in random patterns that were indistinguishable from those produced by computer simulations of two-dimensional random motion. The multivalent gold probes had an average lateral diffusion coefficient (D) of 0.26 x 108 cm2/sec, and paucivalent probes had an average D of 0.73 x 10-8 cm2/sec. Sixteen percent of the multivalent and 50% of the paucivalent probes had values for D in excess of 0.6 X 10-8 cm2/sec, which, after allowance for stochastic variation, are consistent with the D of 1.3 X 10-8 cm2/sec measured by fluorescence recovery after photobleaching of Fl-PtdEtn in the planar bilayer. The effect of valency on diffusion suggests that the multivalent gold binds several lipids forming a disk up to 30-40 nm in diameter, resulting in reduced diffusion with respect to the paucivalent gold, which binds one or a very few lipids. Provided the valency of the gold probe is considered in the interpretation of the results, Nanovid microscopy is a valid method for analyzing the movements of single or small groups of molecules within membranes.Nanometer-size colloidal gold probes combined with videoenhanced microscopy (nanovid microscopy) is a useful tool for studying the movements of proteins within the plasma membrane of living cells (1-7). For example, the value of nanovid microscopy already has been demonstrated for examining putative flow and transport in locomoting cells (2-7). In addition, the possibility of following the movements of individual membrane molecules has unique potential for studying the existence and size of domains in the plasma membrane (3,4,8). An important question for these new probes of motion is whether the attachment of the colloidal gold particle alters the diffusion characteristics of the bound membrane molecule. When compared with values obtained by fluorescence recovery after photobleaching (FRAP), the lateral diffusion coefficient is lower for gold-Con A on macrophages (4) but not for gold-anti-2A1-A on growth cones (5). In some instances, steric hindrance to the movements of the gold molecule could be produced by the glycocalyx, which can be as much as 50-nm thick on some cell types (9).Additionally, motion may be affected by the number of antigen binding sites on the gold probe and its degree of aggregation.In this study we characterize the effect of the colloidal gold probe on Brownian motion of memb...

Fluidic supramolecular nano- and microfibres as molecular rails for regulated movement of nanosubstances

“…Th ese values are almost one order of magnitude lower than those for free spheres in water, calculated on the basis of the Stokes -Einstein equation. Moreover, these values are comparable with those for 30 nm nanogold-coated anti-fl ourescein (0.26 μ m 2 s − 1 ), which was attached to fl uorescein-conjugated phosphatidylethanolamine in a supported bilayer membrane 45 , and vesicles (30 -200 nm in diameter, typically 0.2 μ m 2 s − 1 ) attached to a DNA -lipid conjugate by DNA hybridization in a supported bilayer membrane 46 . Th e latter particles showed two-dimensional random motion on the substrates, which was independent of vesicle size, because of the fl uidity of the membrane.…”

Lipid-Mediated Endocytosis

“…The lateral mobility of GSL-bound polyomaviridae is even lower Kukura et al 2009). Why the diffusion of lipid-bound ligands is reduced to such an extent is not clear, especially because the binding of antibody to lipids does not result in a similar reduction (Lee et al 1991). Interestingly, the binding of cholera toxin to GM1 strongly reduces the diffusion of other lipid molecules in the same membrane (Forstner et al 2006).…”

Control of Immune Responses by Trafficking Cell Surface Proteins, Vesicles and Lipid Rafts to and from the Immunological Synapse

“…This is not the case for single particle tracking (SPT) where the trajectory of individual surface molecules can be studied. In SPT, for example, a gold bead or a quantum dot is attached to a molecule on the cell surface (116,117); the position of the probe can be determined with down to 5-10 nm accuracy (117). By looking at individual molecules rather than ensembles of molecules a hopping behaviour has been revealed and related to transient confinement zones existing in the membrane (60).…”