Characterization of the diffusion of epidermal growth factor receptor clusters by single particle tracking.

A number of studies have shown that receptors of the epidermal growth factor receptor family (ErbBs) exist as higher-order oligomers (clusters) in cell membranes in addition to their monomeric and dimeric forms. Characterizing the lateral diffusion of such clusters may provide insights into their dynamics and help elucidate their functional relevance. To that end, we used single particle tracking to study the diffusion of clusters of the epidermal growth factor (EGF) receptor (EGFR; ErbB1) containing bound fluorescently-labeled ligand, EGF. EGFR clusters had a median diffusivity of 6.8×10(-11)cm(2)/s and were found to exhibit different modes of transport (immobile, simple, confined, and directed) similar to that previously reported for single EGFR molecules. Disruption of actin filaments increased the median diffusivity of EGFR clusters to 10.3×10(-11)cm(2)/s, while preserving the different modes of diffusion. Interestingly, disruption of microtubules rendered EGFR clusters nearly immobile. Our data suggests that microtubules may play an important role in the diffusion of EGFR clusters either directly or perhaps indirectly via other mechanisms. To our knowledge, this is the first report probing the effect of the cytoskeleton on the diffusion of EGFR clusters in the membranes of live cells.

Influence of chain length on the diffusion and electrophoresis of DNA adsorbed on heterogeneous supported lipid bilayers.

This manuscript describes the influence of chain length on the diffusion and electrophoresis of single stranded DNA (ssDNA) adsorbed on heterogeneous cationic supported lipid bilayers. These studies are motivated by the increasing interest in developing novel strategies for the separation of DNA. We studied ssDNA molecules with the number of bases (N) varying from 21 to 84. Fluorescence recovery after photobleaching (FRAP) studies revealed that the diffusivity (D) of adsorbed ssDNA varied with N as D approximately N(-1), similar to a trend previously observed for the diffusion of double stranded DNA on homogeneous supported lipid bilayers. In contrast, the electrophoretic mobility of the adsorbed ssDNA in the presence of an applied tangential electric field was independent of N. Our studies indicated that the motion of ssDNA in the presence of an applied electric field was primarily due to electrophoresis and was not influenced significantly by electro-osmotic flow. Our results also suggest that the use of asymmetric diffusion barriers or other tunable obstacles may assist DNA separation on supported lipid bilayers.

Controlling DNA adsorption and diffusion on lipid bilayers by the formation of lipid domains.

We describe the influence of membrane heterogeneity on the adsorption and diffusion of DNA. Cellular membranes are believed to contain domains (lipid rafts) that influence processes ranging from signal transduction to the diffusion of membrane components. By analogy, we demonstrate that the formation of raft-like domains in supported lipid bilayers provides control over the adsorption and diffusion of DNA. The formation of bilayers from a mixture of the gel phase zwitterionic lipid 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC) and the fluid phase cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) yielded coexisting DSPC-enriched and DOTAP-enriched phases. We demonstrated the ability to pattern the adsorption of DNA on the heterogeneous bilayers, with the adsorption being restricted to the DOTAP-enriched phase. We further demonstrated that the DSPC-enriched domains acted as obstacles to the lateral diffusion of adsorbed DNA. Fluorescence recovery after photobleaching (FRAP) analysis revealed that the diffusivity of the adsorbed DNA tracked that of the underlying lipid, although the lipid diffusivity changed by an order of magnitude with changes in bilayer composition. Fundamental insight into the adsorption and diffusion of DNA on heterogeneous surfaces may be useful for the design of novel techniques for the size-based separation of DNA.

Enhancing protein stability by adsorption onto raftlike lipid domains.

We demonstrate that the stability of adsorbed proteins can be enhanced by controlling the heterogeneity of the surfaceby creating raftlike domains in a soft liposomal membrane. Recent work has shown that enzymes adsorbed onto highly curved nanoscale supports can be more stable than those adsorbed on flat surfaces with nominally the same chemical structure. This effect has been attributed to a decrease in lateral interenzyme interactions on a curved surface. Exploiting this idea, we asked if adsorbing enzymes onto "patchy" surfaces composed of adsorbing and nonadsorbing regions can be used to reduce lateral interactions even on relatively flat surfaces. We demonstrate that creating domains on which an enzyme can adsorb enhances the stability of that enzyme under denaturing conditions. Furthermore, we demonstrate that the size of these domains has a considerable effect on the degree of stability imparted by adsorption. Such biomimetic raft-inspired systems may find use in applications ranging from biorecognition to the design of novel strategies for the separation of biomolecules and controlling the interaction of multicomponent membrane-bound enzymes.