The degree of domain registration in a liquid-ordered/liquid-disordered phase-separating lipid mixture consisting of 1-stearoyl-2-oleoyl-sn-3-phosphocholine, egg sphingomyelin, and cholesterol (molar mixing ratio of 1:1:1) was studied using three different planar lipid bilayer architectures distinguished by their bilayer-substrate distance d using epifluorescence microscopy. The bilayer systems, which were built layer by layer using Langmuir-Blodgett/Schaefer film depositions, included a solid-supported bilayer (d approximately 15 A) and two polymer-supported bilayers with d approximately 30 A and d approximately 58 A, respectively. Complete domain registration between Langmuir-Blodgett and Schaefer monolayer domains was observed for d approximately 58 A but not in the cases when d approximately 15 A and d approximately 30 A. Building the bilayer layer by layer guaranteed that any preexisting domains were not in registration initially; our data show that the domain registration observed was not caused by lipid flip-flop or by lateral rearrangement of preexisting large-scale domains. Instead, additional studies on bilayer systems with asymmetric lipid composition indicate that preexisting domains in the Langmuir-Blodgett monolayer induce the formation of completely registered domains in the opposite Schaefer monolayer. This study provides insight into possible biophysical mechanisms of transbilayer domain coupling. Our findings support the concept that the formation of transbilayer signaling platforms based on registered raft domains may occur without the active involvement of membrane-spanning proteins.

The goal of this study was to increase in vivo microbubble circulation persistence for applications in medical imaging and targeted drug delivery. Our approach was to investigate the effect of lipid monolayer in-plane rigidity to reduce the rate of microbubble dissolution, while holding constant the microbubble size, concentration and surface architecture. We first estimated the impact of acyl chain length of the main diacyl phosphatidyl-choline (PC) lipid and inter-lipid distance on the cohesive surface energy and, based on these results, we hypothesized that microbubble stability and in vivo ultrasound contrast persistence would increase monotonically with increasing acyl chain length. We therefore measured microbubble in vitro stability to dilution with and without ultrasound exposure, as well as in vivo ultrasound contrast persistence. All measurements showed a sharp rise in stability between DPPC (C16:0) and DSPC (C18:0), which correlates to the wrinkling transition, which signals the onset of significant surface shear and gas permeation resistance, observed in prior single-bubble dissolution studies. Further evidence for the effect of the wrinkling transition came from an in vitro and in vivo stability comparison of microbubbles coated with pure DPPC with those of lung surfactant extract. Microbubble stability against dilution without ultrasound and in vivo ultrasound contrast persistence showed a monotonic increase with acyl chain length from DSPC to DBPC (C22:0). However, we also observed that stability dropped precipitously for all measurements on further increasing lipid acyl chain length from DBPC to DLiPC (C24:0). This result suggests that hydrophobic mismatch between the main PC lipid and the lipopolymer emulsifier, DSPE-PEG5000, may drive a less stable surface microstructure. Overall, these results support our general hypothesis of the role of in-plane rigidity for increasing the lifetime of microbubble circulation.

Proper cholesterol transport is essential to healthy cellular activity and any abnormality can lead to several fatal diseases. However, complete understandings of cholesterol homeostasis in the cell remains elusive, partly due to the wide variability in reported values for intra- and intermembrane cholesterol transport rates. Here, we used time-resolved small-angle neutron scattering to measure cholesterol intermembrane exchange and intramembrane flipping rates, in situ, without recourse to any external fields or compounds. We found significantly slower transport kinetics than reported by previous studies, particularly for intramembrane flipping where our measured rates are several orders of magnitude slower. We unambiguously demonstrate that the presence of chemical tags and extraneous compounds employed in traditional kinetic measurements dramatically affect the system thermodynamics, accelerating cholesterol transport rates by an order of magnitude. To our knowledge, this work provides new insights into cholesterol transport process disorders, and challenges many of the underlying assumptions used in most cholesterol transport studies to date.

Distinct lipid environments, including lipid rafts, are increasingly recognized as a crucial factor affecting membrane protein function in plasma membranes. Unfortunately, an understanding of their role in membrane protein activation and oligomerization has remained elusive due to the challenge of characterizing these often small and transient plasma membrane heterogeneities in live cells. To address this difficulty, we present an experimental model membrane platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains (type I) and homogeneous cholesterol-lipid mixtures (type II) into which transmembrane proteins are incorporated (α(v)β(3) and α(5)β(1) integrins). These flexible lipid platforms enable the use of confocal fluorescence spectroscopy, including the photon counting histogram method, in tandem with epifluorescence microscopy to quantitatively probe the effect of the binding of native ligands from the extracellular matrix ligands (vitronectin and fibronectin for α(v)β(3) and α(5)β(1), respectively) on domain-specific protein sequestration and on protein oligomerization state. We found that both α(v)β(3) and α(5)β(1) sequester preferentially to nonraft domains in the absence of extracellular matrix ligands, but upon ligand addition, α(v)β(3) sequesters strongly into raft-like domains and α(5)β(1) loses preference for either raft-like or nonraft-like domains. A corresponding photon counting histogram analysis showed that integrins exist predominantly in a monomeric state. No change was detected in oligomerization state upon ligand binding in either type I or type II bilayers, but a moderate increase in oligomerization state was observed for increasing concentrations of cholesterol. The combined findings suggest a mechanism in which changes in integrin sequestering are caused by ligand-induced changes in integrin conformation and/or dynamics that affect integrin-lipid interactions without altering the integrin oligomerization state.

The current study reports on the inter-leaflet coupling of obstructed lipid diffusion in a polymertethered phospholipid bilayer, where the obstruction of diffusion is caused by lipopolymers which form non-bilayer-spanning membrane pinning sites in the bottom leaflet of the bilayer. Monolayer-specific wide-field single molecule fluorescence microscopy experiments of fluorescence-tagged phospholipids (TRITC-DHPE) over a wide range of lipopolymer concentrations, c tether , reveal a strong, polymerinduced inter-leaflet coupling of obstructed lipid diffusion for different types of lipopolymers, including those based on poly(ethylene glycol), poly(2-methyl-2-oxazoline), and poly(2-ethyl-2-oxazoline) as the hydrophilic polymer moiety. Remarkably, the degree of inter-leaflet diffusional coupling can be regulated by the cholesterol (CHOL) content which affects membrane bending elasticity. This latter finding suggests that the inter-leaflet coupling of obstructed diffusion is caused by polymer-induced bilayer deformations around membrane pinning sites, thus creating membrane-spanning regions of high membrane tension. Because the inter-leaflet coupling of obstructed diffusion at an elevated CHOL molar concentration also decreases with increasing c tether , we hypothesize that both leaflets of the bilayer are morphologically decoupled at high c tether with the outer (lipopolymer-free) monolayer being flatter than the inner one. Our findings could be of biological relevance because a similar mechanism of transbilayer coupling of obstructed diffusion may occur in some regions of cellular membranes.