Lateral Sorting Research Articles

Energetics of the Transmembrane Peptide Sorting by Hydrophobic Mismatch.

Hydrophobic mismatch between a lipid membrane and embedded transmembrane peptides or proteins plays a role in their lateral localization and function. Earlier studies have resolved numerous mechanisms through which the peptides and membrane proteins adapt to mismatch, yet the energetics of lateral sorting due to hydrophobic mismatch have remained elusive due to the lack of suitable computational or experimental protocols. Here, we pioneer a molecular dynamics simulation approach to study the sorting of peptides along a membrane thickness gradient. Peptides of different lengths tilt and diffuse along the membrane to eliminate mismatch with a rate directly proportional to the magnitude of mismatch. We extract the 2-dimensional free energy profiles as a function of local thickness and peptide orientation, revealing the relative contributions of sorting and tilting, and suggesting their thermally accessible regimes. Our approach can readily be applied to study other membrane systems of biological interest where hydrophobic mismatch, or membrane thickness in general, plays a role.

Characteristic lengths of transmembrane peptides controlling their tilt and lateral distribution between membrane domains.

Lipids and proteins of plasma membranes of eukaryotic cells are supposed to form protein-lipid domains, characterized by a different molecular order, bilayer thickness, and elastic parameters. Several mechanisms of preferable distribution of transmembrane proteins to the ordered or disordered membrane domains have been revealed. The mismatch between the length of the protein transmembrane domain and hydrophobic thickness of the lipid bilayer is considered to be an important driving force of protein lateral sorting. Utilizing the continuum theory of elasticity, we analyzed optimal configurations and preferable membrane domains for single-pass transmembrane peptides of various hydrophobic lengths and effective molecular shapes. We obtained that short transmembrane peptides stand perpendicularly to the membrane plane. The exceedance of a certain characteristic length leads to the tilt of the peptide. This length depends on the bilayer thickness. Thus, in the membrane with coexisting ordered (thicker) and disordered (thinner) phases tilting of the peptide in each phase is governed by its individual characteristic length. The lateral distribution of the peptides between ordered and disordered membrane domains is shown to be described by two additional characteristic lengths. The exceedance of the smaller one drives the peptide towards a more ordered and thicker membrane, while the exceedance of the larger characteristic length switches the preferable membrane domain from ordered and thicker to disordered and thinner. Thus, membrane proteins with long enough transmembrane domains are predicted to accumulate in the thinner disordered membrane as compared to the thicker ordered bilayer. For hourglass-like and barrel-like shaped transmembrane peptides the specific regime of sorting was obtained: the peptides distributed almost equally between the phases in a wide range of peptide lengths. This finding allowed explaining the experimental data on lateral distribution of transmembrane peptide tLAT.

Modulation of E-Cadherin and N-Cadherin by ovarian steroids and embryonic stimuli

Endometrium is a dynamic tissue that undergoes extensive remodelling to attain a receptive state which is further modulated in presence of an embryo for successful initiation of pregnancy. Cadherins are the proteins of the junctional complex of which E-cadherin (E-Cad) is crucial for maintaining epithelial cell state and integrity of the epithelial barrier; gain of N-cadherin (N-Cad) in epithelial cells leads to epithelial to mesenchymal transition (EMT). In the present study, we investigated the expression of E-Cad and N-Cad in the mouse endometrial luminal epithelium and its modulation by estrogen, progesterone, and embryonic stimuli. We observed that E-Cad is diffusely expressed in the luminal epithelium of mouse endometrium during the estrus stage and upon estrogen treatment. It is apico-laterally and basolaterally sorted at the diestrus stage and in response to the combined treatment of estrogen and progesterone. In 3D spheroids of human endometrial epithelial cells, combined treatment with estrogen and progesterone led to lateral sorting of E-Cad without any effects on its mRNA levels. at the time of embryo implantation, there is loss of E-Cad along with the gain of N-Cad and SNAIL expression suggestive of EMT in the luminal epithelium. This EMT is possibly driven by embryonic stimuli as treatment with estrogen and progesterone did not lead to the gain of N-Cad expression in the mouse endometrium in vivo or in human endometrial epithelial cells in vitro. In conclusion, the present study demonstrates that steroid hormones directly affect E-Cad sorting in the endometrial epithelium which undergo EMT in response to embryonic stimuli.

Serotonin Alters the Phase Equilibrium of a Ternary Mixture of Phospholipids and Cholesterol.

Unsaturated and saturated phospholipids tend to laterally segregate, especially in the presence of cholesterol. Small molecules such as neurotransmitters, toxins, drugs etc. possibly modulate this lateral segregation. The small aromatic neurotransmitter serotonin (5-HT) has been found to bind to membranes. We studied the lipid structure and packing of a ternary membrane mixture consisting of palmitoyl-oleoyl-phosphatidylcholine, palmitoyl-sphingomyelin, and cholesterol at a molar ratio of 4/4/2 in the absence and in the presence of 5-HT, using a combination of solid-state 2H NMR, atomic force microscopy, and atomistic molecular dynamics (MD) simulations. Both NMR and MD report formation of a liquid ordered (Lo) and a liquid disordered (Ld) phase coexistence with small domains. Lipid exchange between the domains was fast such that single component 2H NMR spectra are detected over a wide temperature range. A drastic restructuring of the domains was induced when 5-HT is added to the membranes at a 9 mol% concentration relative to the lipids. 2H NMR spectra of all components of the mixture showed two prominent contributions indicative of molecules of the same kind residing both in the disordered and the ordered phase. Compared to the data in the absence of 5-HT, the lipid chain order in the disordered phase was further decreased in the presence of 5-HT. Likewise, addition of serotonin increased lipid chain order within the ordered phase. These characteristic lipid chain order changes were confirmed by MD simulations. The 5-HT-induced larger difference in lipid chain order results in more pronounced differences in the hydrophobic thickness of the individual membrane domains. The correspondingly enlarged hydrophobic mismatch between ordered and disordered phases is assumed to increase the line tension at the domain boundary, which drives the system into formation of larger size domains. These results not only demonstrate that small membrane binding molecules such as neurotransmitters have a profound impact on essential membrane properties. It also suggests a mechanism by which the interaction of small molecules with membranes can influence the function of membrane proteins and non-cognate receptors. Altered membrane properties may modify lateral sorting of membrane protein, membrane protein conformation, and thus influence their function as suspected for neurotransmitters, local anesthetics, and other small drug molecules.

Near-field resonant photon sorting applied: dual-band metasurface quantum well infrared photodetectors for gas sensing

Abstract Two photodetectors for measuring transmission and two bulky, separated narrowband filters for picking a target gas absorption line and a non-absorbing reference from broadband emission are typically required for dual-band non-dispersive infrared (NDIR) gas sensing. Metal-dielectric-metal (MDM) metasurface plasmon cavities, precisely controllable narrowband absorbers, suggest a next-generation, nanophotonic approach. Here, we demonstrate a dual-band MDM cavity detector that consolidates the function of two detectors and two filters into a single device by employing resonant photon sorting-a function unique to metasurfaces. Two MDM cavities sandwiching a quantum well infrared photodetector (QWIP) with distinct resonance wavelengths are alternately arranged in a subwavelength period. The large absorption cross section of the cavities ensures ~95% efficient lateral sorting of photons by wavelength into the corresponding detector within a near-field region. The flow of incident photons is thus converted into two independent photocurrents for dual-band detection. Our dual-band photodetectors show competitive external quantum efficiencies up to 38% (responsivity 2.1 A/W, peak wavelength 6.9 5m) at 78 K. By tailoring one resonance to an absorption peak of NO2 (6.25 5m) and the other to a non-absorbing reference wavelength (7.15 5m), NDIR NO2 gas sensing with 10 ppm accuracy and 1 ms response times is demonstrated. Through experiment and numerical simulation, we confirm near-perfect absorption at the resonant cavity and suppressed absorption at its non-resonant counterpart, characteristic of resonant photon sorting. Dual-band sensing across the mid-infrared should be possible by tailoring the cavities and quantum well to desired wavelengths.

Elastic deformations mediate interaction of the raft boundary with membrane inclusions leading to their effective lateral sorting

Liquid-ordered lipid domains represent a lateral inhomogeneity in cellular membranes. These domains have elastic and physicochemical properties different from those of the surrounding membrane. In particular, their thickness exceeds that of the disordered membrane. Thus, elastic deformations arise at the domain boundary in order to compensate for the thickness mismatch. In equilibrium, the deformations lead to an incomplete register of monolayer ordered domains: the elastic energy is minimal if domains in opposing monolayers lie on the top of each other, and their boundaries are laterally shifted by about 3 nm. This configuration introduces a region, composed of one ordered and one disordered monolayers, with an intermediate bilayer thickness. Besides, a jump in a local monolayer curvature takes place in this intermediate region, concentrating here most of the elastic stress. This region can participate in a lateral sorting of membrane inclusions by offering them an optimal bilayer thickness and local curvature conditions. In the present study, we consider the sorting of deformable lipid inclusions, undeformable peripheral and deeply incorporated peptide inclusions, and undeformable transmembrane inclusions of different molecular geometry. With rare exceptions, all types of inclusions have an affinity to the ordered domain boundary as compared to the bulk phases. The optimal lateral distribution of inclusions allows relaxing the elastic stress at the boundary of domains.

Schmidt-hammer exposure-age dating (SHD) of sorted stripes on Juvflye, Jotunheimen (central South Norway): Morphodynamic and palaeoclimatic implications

Measurements with an electronic Schmidt-hammer (RockSchmidt) were conducted on 23 sites of sorted stripes (periglacial patterned ground) on Juvflye, Jotunheimen (central South Norway). All were located above the current lower limit of alpine permafrost. Performing Schmidt-hammer exposure-age dating (SHD) based on application of a new local age-calibration equation for RRock-values yielded SHD-ages between 7975 ± 370 and 6660 ± 355 years ago, which are closely comparable to results obtained previously from sorted circles at the same location. The age estimates are interpreted as ‘composite’ ages indicative of upfreezing of boulders, lateral sorting, and subsequent stabilisation. Formation of patterned ground essentially ceased with the onset of the regional Holocene Thermal Maximum (HTM). Neither sorted stripe sites at higher altitude, continuously underlain by permafrost during the entire Holocene, nor those at lower altitudes affected by re-aggradation of permafrost in the late Holocene show signs of significant recent morphodynamic activity. Likely explanations for early- to mid-Holocene stabilisation include (1) substantial changes of soil moisture conditions and related thermodynamics within the active layer affecting frost action, (2) loss of fine-grained substrate matrix from the coarse stripes and hence reduced frost susceptibility, and (3) exhaustion of supply of boulders from the fines-dominated areas. Whereas the sorted stripe data set as a whole did not reproduce the altitudinal gradient characteristic of sorted circles on Juvflye, the strength of the relationship between sorted stripe mean RRock-values and altitude increased with declining slope gradient. Although interpretation of SHD-ages for patterned ground remains challenging, this successful application of the electronic Schmidt-hammer, with its increased efficiency and technical improvements over the mechanical Schmidt-hammer, offers considerable potential for future SHD-studies in both morphodynamic and palaeoclimatic contexts.

Interaction between lateral sorting in river bends and vertical sorting in dunes

AbstractSediment is sorted in river bends under the influence of gravity that pulls the heavier grains downslope and secondary flow that drags the finer grains upslope. Furthermore, when dunes are present, sediment is also sorted vertically at the dune lee side. However, sorting functions are poorly defined, since the relation to transverse bed slope and the interaction between lateral and vertical sorting is not yet understood for lack of data under controlled conditions. The objective of this study is to describe lateral sorting as a function of transverse bed slope and to gain an understanding of the interaction between lateral and vertical sorting in river bends. To this end, experiments were conducted with a poorly sorted sediment mixture in a rotating annular flume in which secondary flow intensity can be controlled separately from the main flow velocity, and therefore transverse bed slope towards the inner bend and dune dimensions can be systematically varied. Sediment samples were taken along cross‐sections at the surface of dune troughs and dune crests, and over the entire depth at the location of dune crests (bulk samples), which enabled comparison of the relative contribution of vertical sorting by dunes to lateral sorting by the transverse bed slope. The data show that lateral sorting is always the dominant sorting mechanism in bends, and bulk samples showed minor effects of vertical sorting by dunes as long as all grain‐size fractions are mobile. An empirical bend sorting model was fitted that redistributes the available sediment fractions over the cross‐section as a function of transverse bed slope. Comparison with field data showed that the model accurately reproduces spatially‐averaged trends in sorting at the bend apex in single‐thread channels. The bend sorting model therefore provides a better definition of bend sorting with conservation of mass by size fraction and adds to current understanding of bend sorting. The implication for numerical modelling is that bend sorting mechanisms can be modelled independently of dunes, allowing the application of the active layer concept.

Raman Spectroscopy Study of Curvature-Mediated Lipid Packing and Sorting in Single Lipid Vesicles

Cellular plasma membrane deformability and stability is important in a range of biological processes. Changes in local curvature of the membrane affect the lateral movement of lipids, affecting the biophysical properties of the membrane. An integrated holographic optical tweezers and Raman microscope was used to investigate the effect of curvature gradients induced by optically stretching individual giant unilamellar vesicles (GUVs) on lipid packing and lateral segregation of cholesterol in the bilayer. The spatially resolved Raman analysis enabled detection of induced phase separation and changes in lipid ordering in individual GUVs. Using deuterated cholesterol, the changes in lipid ordering and phase separation were linked to lateral sorting of cholesterol in the stretched GUVs. Stretching the GUVs in the range of elongation factors 1–1.3 led to an overall decrease in cholesterol concentration at the edges compared to the center of stretched GUVs. The Raman spectroscopy results were consistent with a model of the bilayer accounting for cholesterol sorting in both bilayer leaflets, with a compositional asymmetry of 0.63 ± 0.04 in favor of the outer leaflet. The results demonstrate the potential of the integrated holographic optical tweezers-Raman technique to induce deformations to individual lipid vesicles and to simultaneously provide quantitative and spatially resolved molecular information. Future studies can extend to include more realistic models of cell membranes and potentially live cells.

Formation of coarse sediment lags in ice-sediment mixtures: A geomorphic signature of sublimation on regolith surfaces

In many high-latitude locations on Mars, boulder sized clasts are present at the ground surface atop finer-grained sediments. These areas are concentrated in the latitude dependent mantle (LDM), an ice-rich surface unit that is globally distributed polewards of ∼30° latitude. Here, we test the hypothesis that sublimation of buried ice can drive grain size sorting in clastic deposits. We conducted pilot laboratory experiments to determine how buried and interstitial dry ice (CO2) sublimation modifies the grain size distribution in overlying and intermingled lithic sediments. We found that sublimation can sort grains into horizons with distinct grain size distributions by allowing fines to winnow into space freed by sublimation of underlying ice. On sloped surfaces, this mechanism enables lateral sorting by concentrating large grains on the surface and allowing them to slide or roll downslope. We infer that sublimation-driven sorting may affect the surface grain size distribution of solar system objects for which solid-vapor phase transitions dominate (e.g., modern Mars, comet 67P/Churyumov-Gerasimenko, asteroids, etc.).

The use of terrestrial laser scanning for the characterization of a cliff-talus system in the Thompson River Valley, British Columbia, Canada

A postglacial river terrace along the Thompson River in Interior British Columbia, Canada has been monitored using terrestrial laser scanning (TLS) and high-resolution photography for almost a 3-year study to observe the deformation and failure processes, which result in changes in the slope morphology. Change detection using Multiscale Model to Model Cloud Comparison (M3C2) and a multi-scale dimensionality analysis (CANUPO) were performed on the 3-dimensional point cloud data to track the deposition patterns occurring in this active cliff talus system. Changes documented in the analysis of TLS data were verified using the high-resolution photography. Over 1.5 m of valley parallel retreat was captured in a section of the cliff face related to instability of a cobble and boulder horizon beneath a thick fluvial gravel unit. Because of the high-resolution remote sensing data, it was possible to observe a longitudinal sorting of grain sizes (i.e. fall sorting) in this cliff-talus system, whereby the size of individual particles controls the position on the slope. The overall mapped distribution of particle sizes on the slope remained constant for the almost 3-year study period. Flows of granular debris were observed in TLS change detection and the CANUPO analysis was able to display the longitudinal and lateral sorting of grain sizes that occurs during flow. This case history demonstrates that high resolution remote sensing data of large slopes permits us to link the geomorphic processes occurring in the cliff face with mass movement and deposition occurring on the talus slope below.

Reconfigurable, graphene-coated, chalcogenide nanowires with a sub-10-nm enantioselective sorting capability

Chiral surface plasmon polaritons (SPPs) produced by plasmonic nanowires can be used to enhance molecular spectroscopy for biosensing applications. Nevertheless, the switchable stereoselectivity and detection of various analytes are limited by a lack of switchable, chiral SPPs. Using both finite-element method simulations and analytic calculations, we present a graphene-coated chalcogenide (GCC) nanowire that produces mid-infrared, chiral SPPs. The chiral SPPs can be reversibly switched between “on” (transparent) and “off” (opaque) by non-volatile structural state transitions in the dielectric constants of the chalcogenide glass Ge2Sb2Te5. Furthermore, by controlling the Fermi energy of the graphene-coating layer, the nanowire can output either non-chiral or chiral SPPs. A thermal-electric model was built to illustrate the possibility of ultrafast on/off switching of the SPPs at the terminus of the nanowire. Finally, we show that a selective, lateral sorting of sub-10-nm enantiomers can be achieved via the GCC nanowire. Chiral nanoparticles with opposite handedness experience transverse forces that differ in both their sign and magnitude. Our design may pave the way for plasmonic nanowire networks and tunable nanophotonic devices, which require the ultrafast switching of SPPs, and provide a possible approach for a compact, enantiopure synthesis.

About Chemical Strategies to Fabricate Cell-Instructive Biointerfaces with Static and Dynamic Complexity.

Properly functioning cell-instructive biointerfaces are critical for healthy integration of biomedical devices in the body and serve as decisive tools for the advancement of our understanding of fundamental cell biological phenomena. Studies are reviewed that use covalent chemistries to fabricate cell-instructive biointerfaces. These types of biointerfaces typically result in a static presentation of predefined cell-instructive cues. Chemically defined, but dynamic cell-instructive biointerfaces introduce spatiotemporal control over cell-instructive cues and present another type of biointerface, which promises a more biomimetic way to guide cell behavior. Therefore, strategies that offer control over the lateral sorting of ligands, the availability and molecular structure of bioactive ligands, and strategies that offer the ability to induce physical, chemical and mechanical changes in situ are reviewed. Specific attention is paid to state-of-the-art studies on dynamic, cell-instructive 3D materials. Future work is expected to further deepen our understanding of molecular and cellular biological processes investigating cell-type specific responses and the translational steps toward targeted in vivo applications.

Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins.

Lipid rafts are microdomains present in the membrane of eukaryotic organisms and bacterial pathogens. They are characterized by having tightly packed lipids and a subset of specific proteins. Lipid rafts are associated with a variety of important biological processes including signaling and lateral sorting of proteins. To determine whether lipid rafts exist in the inner membrane of Borrelia burgdorferi, we separated the inner and outer membranes and analyzed the lipid constituents present in each membrane fraction. We found that both the inner and outer membranes have cholesterol and cholesterol glycolipids. Fluorescence anisotropy and FRET showed that lipids from both membranes can form rafts but have different abilities to do so. The analysis of the biochemically defined proteome of lipid rafts from the inner membrane revealed a diverse set of proteins, different from those associated with the outer membrane, with functions in protein trafficking, chemotaxis and signaling.

Single-Lipids Diffusion and Lipid Sorting at Nanoscale Curvature Sites

Membrane curvature may cause changes to the local effective membrane viscosity and induce a lateral sorting of membrane components. However, the relationships between curvature, diffusion, and lipid shape have not been established. We used single-particle tracking to observe fluorescently labeled lipids on nanoscale curvature sites in supported lipid bilayers (SLBs). Giant unilamellar vesicles (GUV) were burst over 100 nm diameter nanoparticles (NP) on glass coverslips. Membrane composition variations yielded minimal changes to the effects of curvature, including mixtures of POPC, lysoPC, DOPC, and DLPC. However, membrane curvature had a widely varying effect on different fluorescent lipids. Lipid head labeled TexasRed-DHPE diffused half of the speed on curvature site compared to lipid tail labeled TopFluorPC or TopFluorPIP2 yet two time faster in flat lipid bilayer. The curvature affected these fluorescent lipids in drastically different ways. We hypothesize that diffuser tail properties affects diffusion at curvature sites more than head groups. Further head-labeled lipids and tail-labeled lipids will be studied to examine the generality of this observation. Additionally, the site of the fluorescent label affected the sorting of the fluorescently labeled lipid to the curvature site. Understanding and comparing the molecular sorting in lipid bilayers can further our understanding of the curvature sensing lipids properties such as tail saturate/ unsaturated preference and head group types. These studies will contribute to the greater biophysical understanding of membrane curvature.

Lateral sorting of chiral nanoparticles using Fano-enhanced chiral force in visible region.

Chiral gradient force allows a passive separation of an enantiomer since its direction is dependent on the handedness of its chiral entities. However, chiral polarisability is much weaker than electric polarisability. As a consequence, the non-chiral gradient force dominates over chiral force, which makes enantioselective sorting challenging. We present here, both numerically and analytically, that the chiral gradient force acting on chiral nanoparticles can overcome the non-chiral force when specimens are placed in a Fano-enhanced chiral gradient near-field using a plasmonic nanoaperture. Under circularly polarized light illumination, the interaction between the resonant modes of symmetric outer and asymmetric inner Au split-rings results in a splitting of the modal energies, which excites multipolar interference Fano resonances (FRs). This enables a local aperture between the two split-rings to possess very large optical chirality gradients while maintaining low gradients of electromagnetic energy density around the FRs from the visible region. By way of the lateral resultant force composed of both chiral and non-chiral gradient forces, we can accomplish a helicity-dependent transverse deflection of the chiral nanoparticles positioned above the aperture, which may offer a good platform for all-optical enantiopure compounds.

Modulation of cell surface transport and lipid raft localization by the cytoplasmic tail of the influenza virus hemagglutinin.

SummaryViral glycoproteins are highly variable in their primary structure, but on the other hand feature a high functional conservation to fulfil their versatile tasks during the pathogenic life cycle. Typically, all protein domains are optimized in that indispensable functions can be assigned to small conserved motifs or even individual amino acids. The cytoplasmic tail of many viral spike proteins, although of particular relevance for the virus biology, is often only insufficiently characterized. Hemagglutinin (HA), the receptor‐binding protein of the influenza virus comprises a short cytoplasmic tail of 13 amino acids that exhibits three highly conserved palmitoylation sites. However, the particular importance of these modifications and the tail in general for intracellular trafficking and lateral membrane organization remains elusive. In this study, we generated HA core proteins consisting of transmembrane domain, cytoplasmic tail and a minor part of the ectodomain, tagged with a yellow fluorescent protein. Different mutation and truncation variants of these chimeric proteins were investigated using confocal microscopy, to characterize the role of cytoplasmic tail and palmitoylation for the intracellular trafficking to plasma membrane and Golgi apparatus. In addition, we assessed raft partitioning of the variants by Foerster resonance energy transfer with an established raft marker.We revealed a substantial influence of the cytoplasmic tail length on the intracellular distribution and surface exposure of the proteins. A complete removal of the tail hampers a physiological trafficking of the protein, whereas a partial truncation can be compensated by cytoplasmic palmitoylations. Plasma membrane raft partitioning on the other hand was found to imperatively require palmitoylations, and the cysteine at position 551 turned out to be of most relevance.Our data shed further light on the tight interconnection between cytoplasmic elements and intracellular trafficking and suggest a function of HA palmitoylations in both lateral sorting and anterograde trafficking of the glycoprotein.

Resolving Nanoscale Curvature on Lipid Bilayers with Polarized Localization Microscopy

The lateral sorting of membrane lipids and proteins in conjunction with membrane curvature, are postulated to provide a physical basis to initiate and regulate many complex cellular processes such as endocytosis/exocytosis. However, many hypotheses concerning these processes are unanswered because of the diffraction-limited resolution of most optical techniques (∼200 nm). In addition, present imaging techniques are incapable of observing dynamic nanoscale membrane bending and the reorganization of membrane bound molecules around the curvature site. To overcome these experimental limitations, we aim to detect and resolve biological processes involving nanoscale cell membrane curvature by Polarized Localization Microscopy (PLM). Selective excitation of fluorophores embedded in the lipid membrane by p- or s-polarized light reveal distinct vertical versus lateral membrane regions with 18 nm localization uncertainty. To exploit PLM experimental capabilities and improved resolution over current imaging techniques, PLM was performed on systems with induced curvature. For instance, small unilaminar lipid vesicles (50 -/+ 20 nm diameter) provide regions of curvature bound to a supported lipid bilayer (SLBs) with a lipid composition of POPC and 0.3% DiI. Additional samples include SLBs on polymerized large unilaminar vesicles (100 nm -/+ 20 diameter) that induces membrane deformation. The novel technique of PLM detects the presence of the vesicles and resolves their sizes and lateral positions in a super-resolution image. Further, we aim to utilize PLM to investigate the interplay between membrane orientations and nanoscale membrane lipid phase dynamics. Moreover, we anticipate to demonstrate the effect of various membrane orientations on the redistribution of trans-membrane molecules and proteins sorting. These studies will provide fundamental insights of curvature sensitive biological mechanisms that have been previously intractable, including neuronal communication, immunological signaling, and viral infections.

Simple applications of microparticle transportation by tender optical scattering force

This paper provides a novel application of the optical radiation pressure for microfluidic particle transportation without precise focusing or alignment of the laser beam to the device and target. An optical manipulation of particles in a microfluidic platform is highly exploited in life science or biomedical analysis using optical tweezers with the use of a gradient force of the optical radiation pressure. Our method utilizes the other term of the radiation pressure, namely scattering force, to manipulate particles in a microchannel. The migration distance of particle depends on the amount of light received by the particle. Therefore, particle movement with long retention distance can be achieved by large-area irradiation even with low energy density. In our experiments, two proof-of-concept microfluidic chips were designed and investigated; one was a lateral particle sorting using a monolithic microfluidic chip integrated with a planar SU-8 waveguide and beam expander, the other was a vertical sorting using a 10-cm-long polydimethylsiloxane channel with whole-area irradiation. Experimental results show that 1, 2 and 5 μm polystyrene beads can be transported by the optical scattering force and that particle migration is achieved with the irradiated energy density <10 mW/mm2. The present method has practical potential for simple and fuss-free use of the optical radiation pressure without spot focusing or precise alignment process of the laser beam and damage to the device and sample.

The cholesterol-binding motif of the HIV-1 glycoprotein gp41 regulates lateral sorting and oligomerization.

Enveloped viruses often use membrane lipid rafts to assemble and bud, augment infection and spread efficiently. However, the molecular bases and functional consequences of the partitioning of viral glycoproteins into microdomains remain intriguing questions in virus biology. Here, we measured Foerster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) to study the role of distinct membrane proximal regions of the human immunodeficiency virus glycoprotein gp41 for lipid raft partitioning in living Chinese hamster ovary cells (CHO-K1). Gp41 was labelled with a fluorescent protein at the exoplasmic face of the membrane, preventing any interference of the fluorophore with the proposed role of the transmembrane and cytoplasmic domains in lateral organization of gp41. Raft localization was deduced from interaction with an established raft marker, a fluorescently tagged glycophosphatidylinositol anchor and the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial lateral sorting determinant in CHO-K1 cells. Interestingly, the raft association of gp41 indicates a substantial cell-to-cell heterogeneity of the plasma membrane microdomains. In complementary fluorescence polarization microscopy, a distinct CRAC requirement was found for the oligomerization of the gp41 variants. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral sorting of viral glycoproteins for virus assembly at cellular membranes.