There is growing recognition that lipid heterogeneities in cellular membranes play an important role in the distribution and functionality of membrane proteins. However, the detection and characterization of such heterogeneities at the cellular level remains challenging. Here we report on the poorly understood relationship between lipid bilayer asymmetry and membrane protein sequestering in raft-mimicking model membrane mixtures using a powerful experimental platform comprised of confocal spectroscopy XY-scan and photon-counting histogram analyses. This experimental approach is utilized to probe the domain-specific sequestering and oligomerization state of αvβ3 and α5β1 integrins in bilayers, which contain coexisting liquid-disordered/liquid-ordered (ld/lo) phase regions exclusively in the top leaflet of the bilayer (bottom leaflet contains ld phase). Comparison with previously reported integrin sequestering data in bilayer-spanning lo-ld phase separations demonstrates that bilayer asymmetry has a profound influence on αvβ3 and α5β1 sequestering behavior. For example, both integrins sequester preferentially to the lo phase in asymmetric bilayers, but to the ld phase in their symmetric counterparts. Furthermore, our data show that bilayer asymmetry significantly influences the role of native ligands in integrin sequestering.

The current study reports the use of buckling structures as a measure of film elasticity on physisorbed, solid-supported polymer-tethered lipid monolayers consisting of phospholipids and poly(ethylene glycol) (PEG) lipopolymers. These monolayer systems, built using the Langmuir-Blodgett (LB) technique, exhibit buckles over a wide range of lipopolymer concentrations. Systematic quantitative analysis of the buckling structures using epifluorescence microscopy and atomic force microscopy reveals that increased lipopolymer concentrations are associated with higher area fractions of buckling regions. Quantitative analyses of buckling profiles show, furthermore, that enhanced lipopolymer concentrations lead to increased buckling widths without notably altering buckling amplitude. On the basis of these experimentally determined buckling parameters, we are able to derive a metric between elastic properties and buckling structures in the polymer-tethered lipid monolayer system. This is achieved by combining mean-field calculations of elastic properties of polymer-tethered lipid membranes with buckling theory of an Euler column. Our findings are significant because they provide new insight into the fascinating materials properties of polymer-lipid supramolecular assemblies and represent a tool for the characterization of elastic properties in complex biomembrane-mimicking film architectures with buckling structures.

Abstract:The influence of cholesterol concentration on the formation of buckling structures is studied in a physisorbed polymer-tethered lipid monolayer system using epifluorescence microscopy (EPI) and atomic force microscopy (AFM). The monolayer system, built using the Langmuir-Blodgett (LB) technique, consists of 3 mol % poly(ethylene glycol) (PEG) lipopolymers and various concentrations of the phospholipid, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and cholesterol (CHOL). In the absence of CHOL, AFM micrographs show only occasional buckling structures, which is caused by the presence of the lipopolymers in the monolayer. In contrast, a gradual increase of CHOL concentration in the range of 0-40 mol % leads to fascinating film stress relaxation phenomena in the form of enhanced membrane buckling. Buckling structures are moderately deficient in CHOL, but do not cause any notable phospholipid-lipopolymer phase separation. Our experiments demonstrate that membrane buckling in physisorbed polymer-tethered membranes can be controlled through CHOL-mediated adjustment of membrane elastic properties. They further show that CHOL may have a notable impact on molecular confinement in the presence of crowding agents, such as lipopolymers. Our results are significant, because they offer an intriguing prospective on the role of CHOL on the material properties in complex membrane architecture. OPEN ACCESSPolymers 2013, 5 405

have a cholate backbone with polar groups extending from one face and a short alkyl chain extending from the opposite face. FA-solubilized Cx26 crystallized into the H32 space group with two monomers in the asymmetric unit, and the crystals diffracted isotropically to 3.3-Å resolution. Using a molecular replacement search model based on a cryoEM map of Cx43 at 5.7-Å resolution [Fleishman et al., Mol. Cell 15: 879-888 (2004)], we solved the structure independently from a previously reported 3.5-Å resolution X-ray structure [2ZW3, Maeda et al., Nature 458: 597-602 (2009)]. The overall R/Rfree values and completeness were 0.311/0.328 and 98.9%, respectively, and the Molprobity score was 2.07 (100 th percentile). The RMS differences between 2ZW3 and our structure were 0.9 and 1.7-Å for the main-chain and side-chain atoms in the TM helices and 1.3 and 1.9-Å for the main-chain and side-chain atoms in E1 and E2. Although the topology and fold recapitulated 2ZW3, the maximum differences were significant: 2.7 and 5.9-Å for the main chain and side chain atoms in the TM helices and 3.9 and 7.6-Å for the main-chain and side-chain atoms in E1 and E2. We generated an electron density map at a resolution comparable to the cryoEM structure of the authentic Cx43 channel. The similarity of the maps suggests that detergent-solubilized Cx26 that crystallized as a dodecamer represents the authentic gap junction channel.

Quite surprisingly, N-and the C-termini remained in close proximity at high denaturant concentrations for ca. 40% of the conformations, suggesting that DrkN-SH3behaves at least partially like a disordered circular chain.

Thymidine block that arrests cells at the border between G1 and S phases. Critical temperatures were elevated in GPMVs isolated from cells in cell cycle phases that precede cell division (G2 and M) compared to other stages (G1 and S). In unsynchronized cells, critical temperatures were found to be inversely proportional to cell density, suggesting that contact inhibition and associated arrest of cell growth results in lower plasma membrane critical temperatures. Lower critical temperatures were also observed when growth was arrested through overnight serum starvation, and elevated critical temperatures were restored 24h after the addition of serum containing medium. Transition temperatures are also lowered in GPMVs prepared from cells undergoing apoptosis through the application of TRAIL, and vesicles contain a more rigid liquid-ordered or gel phase at low temperature. These results are in agreement with past studies that have indicated that plasma membrane composition varies within the cell cycle. Since GPMV critical temperatures are hypothesized to reflect on the magnitude of lipid-mediated heterogeneity in intact cells, these results suggest that membrane heterogeneity is greatest in cells undergoing rapid growth and cell division and is suppressed in cells under low growth conditions. Nano-membrane domains are hypothesized to play an integral role in many cell signaling pathways. Their transient nature and biocomplexity underlies a myriad of fundamental questions about lipid-lipid and lipid-protein interactions and their roles in cellular functions. As a result, there is a need for innovative approaches for understanding different biophysical aspects of membrane assemblies and their underlying, multiscale dynamics. Here, we integrate dynamic holographic optical trapping (HOT) and fluorescence imaging with fluorescence correlation spectroscopy (FCS) to characterize membrane domain nucleation in biomimetic planar supported bilayers. The dynamic HOT system allows for the creation of multiple traps from a single light source, each of which can be controlled individually in real time. Silica microspheres are being trapped into arbitrary patterns for system optimization. Receptor-bound microspheres associated with nano-domains in planar supported bilayers act as handles for dynamic HOT manipulation. Our hypothesis is that by trapping multiple microsphere-bound receptors, the associated heterogeneous lipid domains will nucleate a larger domain upon interaction in a manner that depends on the lipid type, cholesterol and protein content. Fluorescence imaging is used to visualize lipid domain formation, and subsequent lateral diffusion of lipid species will be measured with FCS as a function of trap-induced confinement. These results will ultimately lead to new insights into domain formation in membranes. It is now well established that the cytoplasm and plasma membrane of cells are characterized by high concentrations of proteins. Consequently, macromolecular crowding and confinement effects are believed to play impor...

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