Proximal Leaflet Research Articles

Leaflet by Leaflet Synergistic Effects of Antimicrobial Peptides on Bacterial and Mammalian Membrane Models.

Antimicrobial peptides (AMPs) offer significant hope in the fight against antibiotic resistance. Operating via a mechanism different from that of antibiotics, they target the microbial membrane and ideally should damage it without impacting mammalian cells. Here, the interactions of two AMPs, magainin 2 and PGLa, and their synergistic effects on bacterial and mammalian membrane models were studied using electrochemical impedance spectroscopy, atomic force microscopy (AFM), and fluorescence correlation spectroscopy. Toroidal pore formation was observed by AFM when the two AMPs were combined, while individually AMP effects were confined to the exterior leaflet of the bacterial membrane analogue. Using microcavity-supported lipid bilayers, the diffusivity of each bilayer leaflet could be studied independently, and we observed that combined, the AMPs penetrate both leaflets of the bacterial model but individually each peptide had a limited impact on the proximal leaflet of the bacterial model. The impact of AMPs on a ternary, mammalian mimetic membrane was much weaker.

Inter-Leaflet Phospholipid Exchange Impacts the Ligand Density Available for Protein Binding at Supported Lipid Bilayers.

Phospholipid bilayers formed at solid-liquid interfaces have garnered interest as mimics of cell membranes to model association reactions of proteins with lipid bilayer-tethered ligands. Despite the importance of understanding how ligand density in a lipid bilayer impacts the protein-ligand association response, relating the ligand-modified lipid fraction to the absolute density of solution-accessible ligands in a lipid bilayer remains a challenge in interfacial quantitative analysis. In this work, confocal Raman microscopy is employed to quantify the association of anti-biotin IgG with a small fraction of biotinylated lipids dispersed in either gel-phase or liquid-crystalline supported lipid bilayers deposited on the interior surfaces of wide-pore silica surfaces. We examine the question of whether inter-leaflet lipid translocation contributes to the population of solution-accessible biotin ligands on the distal leaflet of a supported lipid bilayer by comparing their protein accumulation response with ligands dispersed in lipid monolayers on nitrile-derivatized silica surfaces. The binding of the antibody to biotin ligands dispersed in gel-phase bilayers exhibited an equivalent biotin coverage response as the accumulation of IgG onto gel-phase monolayers, indicating that gel-phase bilayer symmetry was preserved. This result contrasts with the ∼60% greater anti-biotin capture observed at fluid-phase bilayers compared to fluid-phase monolayers prepared at equivalent biotin fractions. This enhanced protein capture is attributed to biotin-capped lipids being transferred from the surface-associated proximal leaflet of the bilayer to the solution-exposed distal leaflet by the inter-leaflet exchange or lipid flip-flop, a facile process in fluid-phase supported lipid bilayers. The results suggest caution in interpreting the results of quantitative studies of protein binding to lipid-tethered ligands dispersed in fluid-phase phospholipid bilayers.

Developmental Analysis of Compound Leaf Development in Arachis hypogaea.

Leaves are the primary photosynthetic structures, while photosynthesis is the direct motivation of crop yield formation. As a legume plant, peanut (Arachis hypogaea) is one of the most economically essential crops as well as an important source of edible oil and protein. The leaves of A. hypogaea are in the tetrafoliate form, which is different from the trifoliate leaf pattern of Medicago truncatula, a model legume species. In A. hypogaea, an even-pinnate leaf with a pair of proximal and distal leaflets was developed; however, only a single terminal leaflet and a pair of lateral leaflets were formed in the odd-pinnate leaf in M. truncatula. In this study, the development of compound leaf in A. hypogaea was investigated. Transcriptomic profiles revealed that the common and unique differentially expressed genes were identified in a proximal leaflet and a distal leaflet, which provided a research route to understand the leaf development in A. hypogaea. Then, a naturally occurring mutant line with leaf developmental defects in A. hypogaea was obtained, which displayed a pentafoliate form with an extra terminal leaflet. The characterization of the mutant indicated that cytokinin and class I KNOTTED-LIKE HOMEOBOX were involved in the control of compound leaf pattern in A. hypogaea. These results expand our knowledge and provide insights into the molecular mechanism underlying the formation of different compound leaf patterns among species.

Capsular Closure Following Hip Arthroscopy for Femoroacetabular Impingement.

Capsular closure of the hip occurs following standard hip arthroscopy, which typically includes labral repair and osteoplasty of the femoral neck and/or acetabulum accessed through an interportal capsulotomy. Viewing through an anterolateral portal with use of a 70° scope, a suture-passing device loaded with a #2 FiberWire (Arthrex) is passed down into the mid-anterior portal. The suture is then passed through the anterior aspect of the proximal leaflet of the capsulotomy. The suture-passing device is then passed through the distal leaflet, and the previously passed suture is grasped and brought out of the cannula. The capsular bites are typically made 1 cm from the edge of the capsule. This process is repeated 1.5 cm posteriorly to the initially placed suture. Typical capsular closure takes 2 to 3 sutures for complete closure. Once the sutures are passed, they are tied with use of half-hitches and excess suture is cut. Alternatives include capsular plication, which is more frequently performed in the setting of capsular laxity preoperatively or connective-tissue disorders5,6. Additionally, there is evidence suggesting that leaving the capsulotomy unrepaired may lead to similar results in select populations7. This procedure is performed largely secondary to the restoration of native anatomy and kinematics of the hip joint3,8. Arthroscopy of the hip requires a capsulotomy in order to access the joint, unlike other, more superficial joints such as the shoulder. Capsular closure is often technically challenging and adds length to the procedure. However, recent literature has shown improved patient-reported outcomes with routine capsular closure specifically among high-level athletes9,10. Routine capsular closure has been associated with good mid-term patient-reported outcomes10. Patients who undergo hip arthroscopy and routine capsular closure can expect to meet the minimal clinically important difference and the patient acceptable symptomatic state for the modified Harris hip score and the Hip Outcome Score Activities of Daily Living component. Patients who participate in high-level sports activities may experience a quicker return to play and more normal kinematics with routine capsular closure. Clear off the capsule immediately following the capsulotomy.Utilize an over-the-top view to better visualize the capsule.Place the sutures from anterior to posterior.

Mechanical and Electrical Interaction of Biological Membranes with Nanoparticles and Nanostructured Surfaces.

In this review paper, we theoretically explain the origin of electrostatic interactions between lipid bilayers and charged solid surfaces using a statistical mechanics approach, where the orientational degree of freedom of lipid head groups and the orientational ordering of the water dipoles are considered. Within the modified Langevin Poisson–Boltzmann model of an electric double layer, we derived an analytical expression for the osmotic pressure between the planar zwitterionic lipid bilayer and charged solid planar surface. We also show that the electrostatic interaction between the zwitterionic lipid head groups of the proximal leaflet and the negatively charged solid surface is accompanied with a more perpendicular average orientation of the lipid head-groups. We further highlight the important role of the surfaces’ nanostructured topography in their interactions with biological material. As an example of nanostructured surfaces, we describe the synthesis of TiO2 nanotubular and octahedral surfaces by using the electrochemical anodization method and hydrothermal method, respectively. The physical and chemical properties of these nanostructured surfaces are described in order to elucidate the influence of the surface topography and other physical properties on the behavior of human cells adhered to TiO2 nanostructured surfaces. In the last part of the paper, we theoretically explain the interplay of elastic and adhesive contributions to the adsorption of lipid vesicles on the solid surfaces. We show the numerically predicted shapes of adhered lipid vesicles corresponding to the minimum of the membrane free energy to describe the influence of the vesicle size, bending modulus, and adhesion strength on the adhesion of lipid vesicles on solid charged surfaces.

Leaflet-specific Lipid Diffusion Revealed by Fluorescence Lifetime Correlation Analyses

Cell membranes are fluidic, and the fluidic properties give unique biological functions to the membranes. Here, we review the application of fluorescence lifetime correlation analyses to elucidate the leaflet-specific lipid diffusion in a model cell membrane supported on a glass surface. Utilization of fluorescence lifetime enables us to discriminate the signals from each leaflet of the lipid bilayer and to analyze the lipid diffusion in a leaflet-specific manner. The results showed that the lipid diffusion in a proximal leaflet facing a glass surface is sensitive to the strength of the electrostatic interaction between the lipid headgroup and the glass surface whereas that in the distal one is insensitive to it. This clearly indicates that the interleaflet coupling is negligible. Because the strength of the interleaflet coupling is highly relevant to the cooperative formation of functional domains across the bilayer, this work provides an important insight into the lipid dynamics in cell membranes.

Penetratin translocation mechanism through asymmetric droplet interface bilayers

Penetratin is a cell penetrating peptide (CPP) that can enter cells by direct translocation through the plasma membrane. The molecular mechanism of this translocation still remains poorly understood. Here we provide insights on this mechanism by studying the direct translocation of the peptide across model membranes based on Droplet Interface Bilayers (DIBs), which are bilayers at the interface between two adhering aqueous-in-oil droplets. We first showed with symmetric bilayers made of a mix of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (POPC) that the translocation of penetratin required the presence of at least 40% of POPG on both leaflets. Interestingly when replacing POPG with another anionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), translocation was inefficient. To elucidate the lipid partners required at each step of the CPP translocation process, we then investigated the crossing of asymmetric bilayers. We found that POPG on the proximal leaflet and POPS on the distal leaflet allowed penetratin translocation. Translocation was not observed when POPS was on the proximal leaflet and POPG on the distal leaflet or if POPS on the distal leaflet was replaced with POPC. These observations led us to propose a three-step translocation mechanism: (i) peptide recruitment by anionic lipids, (ii) formation of a transient peptide-lipid structure leading to the initiation of translocation which required specifically POPG on the proximal leaflet, (iii) termination of the translocation process favored by a driving force provided by anionic lipids in the distal leaflet.

Hybrid-Supported Bilayers Formed with Mixed-Charge Surfactants on C18-Functionalized Silica Surfaces.

Mixtures of cationic-anionic surfactants have been shown to spontaneously form ordered monolayers at hydrophobic-hydrophilic boundaries, including air-water and oil-water interfaces. In this work, confocal Raman microscopy is used to investigate the structure of hybrid-supported surfactant bilayers (HSSBs) formed by deposition of a distal leaflet of mixed cationic-anionic surfactants onto a proximal leaflet of n-alkane (C18) chains on the interior surfaces of chromatographic silica particles. The surface coverage of the two surfactants in a hybrid bilayer was determined from carbon analysis and the relative Raman scattering of their respective head-groups. Within the measurement uncertainty, the stoichiometric ratio of the two surfactants is one-to-one, equivalent to mixed-charge-surfactant monolayers at air-water and oil-water interfaces and consistent with the role of the head-group electrostatic interactions in their formation. When self-assembled on the hydrophobic surface, pairs of oppositely charged n-alkyl chain surfactants resemble a phospholipid (phosphatidylcholine) molecule, with its zwitterionic head-group and two hydrophobic acyl chain tails. Indeed, the structure of these hybrid-supported surfactant bilayers on C18-modified silica surfaces is similar to that of hybrid-supported lipid bilayers (HSLBs) on the same supports, but with denser and more-ordered n-alkyl chains. Hybrid-supported surfactant bilayers exhibit a melting phase transition (gel to liquid-crystalline phase) with structural and energetic characteristics similar to those of hybrid-supported bilayers prepared from a zwitterionic phospholipid of the same alkyl chain length. These mixed-charge surfactants on n-alkane-modified silica are stable in water over time (months), results that suggest the potential use of these hybrid bilayers for generating supported lipid-bilayer-like surfaces or for separation applications.

Annexin V Drives Stabilization of Damaged Asymmetric Phospholipid Bilayers.

Annexins are soluble membrane-binding proteins that associate in a calcium dependent manner with anionic phospholipids. They play roles in membrane organization, signaling and vesicle transport and in several disease states including thrombosis and inflammation. Annexin V is believed to be involved in membrane repair. Mediated through binding to phosphatidylserine exposed at damaged plasma membrane, the protein forms crystalline networks that seal or stabilize small membrane tears. Herein, we model this biochemical mechanism to simulate membrane healing at microcavity array supported, transversally asymmetric, lipid bilayers (MSLBs) comprising 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS). Varying annexin V concentration, lipid composition, and DOPS presence at each leaflet, fluorescence imaging and correlation spectroscopy confirmed that when DOPS was present at the external, annexin V, contacting leaflet, the protein assembled rapidly at the membrane interface to form a layer. From electrochemical impedance studies, the annexin layer decreased membrane capacitance while reducing resistance. With DOPS incorporated only at the lower (proximal) leaflet, no appreciable annexin assembly was observed over the first 21 h. This suggests that membrane asymmetry is preserved over this window and transversal diffusion of DOPS is slow. Intense laser light applied to the membrane, in which DOPS is initially isolated at the lower leaflet, was found to simulate membrane damage, stimulating the rapid assembly of annexin V at the membrane interface confirmed by fluorescence imaging, correlation spectroscopy, and electrochemical impedance measurements. The damage induced by light increased impedance and decreased membrane resistance. The resulting bilayer annexin V patched bilayer showed better temporal stability toward impedance changes when compared with that of the parent membrane. In summary, this simple model of annexin V assembly in a fluidic lipid membrane provides new insights into the assembly of annexins as well as an empirical basis for building patch-repair mechanisms into interfacial bilayer membrane assemblies.

Effect of electrostatic interaction on the leaflet-specific diffusion in a supported lipid bilayer revealed by fluorescence lifetime correlation analysis.

A supported lipid bilayer (SLB) is now an indispensable tool to analyze the dynamical properties of biomembranes. However, the effect of a solid support on the leaflet-specific lipid dynamics in a SLB remains elusive, which hampers the further application of the SLB as a model biomembrane. Here, we performed the leaflet-specific lipid diffusion analysis by means of two-dimensional fluorescence lifetime correlation spectroscopy to elucidate the effect of the electrostatic interaction between lipid headgroups and a glass surface on the lipid diffusion in each leaflet of the SLB. The results clearly showed the correlation between the strength of the electrostatic interaction and the lipid diffusion in the proximal leaflet of the SLB facing a glass surface. In particular, the electrostatic attraction between the cationic lipids and a negatively charged glass surface enhanced the lipid diffusion in the proximal leaflet of the SLB, providing important implications for the lipid dynamics not only in the SLB but also in biomembranes.

Reduction of glass-surface charge density slows the lipid diffusion in the proximal leaflet of a supported lipid bilayer.

Understanding the effect of a solid support on the dynamical properties of a supported lipid bilayer (SLB) is a prerequisite for the applications of SLB as a model biomembrane. Here, we applied two-dimensional fluorescence lifetime correlation spectroscopy to examine the effect of solution pH on the diffusion of lipids in the proximal/distal leaflets of a zwitterionic SLB. Leaflet-specific diffusion analyses at various pH revealed that the diffusion of lipids in the proximal leaflet facing a glass surface becomes slower by decreasing pH with the transition pH of ∼7.4. We attributed it to the reduction of the surface charge density of a glass support. Furthermore, the data clearly showed that the lipid diffusion in the distal leaflet facing a bulk solution is insensitive to the change in the diffusion property of the proximal leaflet. This reflects a weak interleaflet coupling between the proximal and distal leaflets of the SLB.

Quantifying the Diffusion of Lipids in the Proximal/Distal Leaflets of a Supported Lipid Bilayer by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy.

A supported lipid bilayer (SLB) is a versatile platform for examining the dynamical properties of biomembranes. However, the effect of a prerequisite solid substrate on the dynamics of a SLB remains very elusive. Especially, it is not clarified how the diffusivity of each leaflet in a SLB is affected by the SLB-solid substrate interaction. In this study, we applied two-dimensional fluorescence lifetime correlation spectroscopy to a SLB for elucidating the diffusion of lipids in the proximal and distal leaflets of a SLB. We find that the autocorrelation curve of a fluorescent lipid in the proximal leaflet decays more slowly than that in the distal leaflet, meaning that the proximal leaflet is less diffusive. This result indicates stronger interaction between the proximal leaflet and a solid substrate.

Electrochemically Triggered Release of Reagent to the Proximal Leaflet of a Microcavity Supported Lipid Bilayer.

A novel and versatile approach to electrichemically triggering the release of a reagent, β-cyclodextrin (β-CD), selectively to the proximal leaflet of a supported lipid bilayer is described. Selective delivery is achieved by creating a spanning lipid bilayer across a microcavity array and exploiting the irreversible redox disassembly of the host-guest complex formed between thiolated ferrocene (Fc) and β-cyclodextrin (β-CD) in the presence of chloride. Self-assembled monolayers of the ferrocene-alkanethiols were formed regioselectively on the interior surface of highly ordered 2.8 μm cavities while the exterior top surface of the array was blocked with a monolayer of mercaptoethanol. The Fc monolayers were complexed with β-CD or β-CD-conjugated to streptavidin (β-CD-SA). Phospholipid bilayers were then assembled across the array via combined Langmuir-Blodgett/vesicle fusion leading to a spanning bilayer suspended across the aqueous filled microcavities. Upon application of a positive potential, ferrocene is oxidized to ferrocinium cation, disrupting the inclusion complex and leading to the release of the β-CD into the microcavity solution where it diffuses to the lower leaflet of the suspended bilayer. Disassembly of the supramolecular complex within the cavities and binding of the β-CD-SA to a biotinylated bilayer was followed by voltammetry and impedance spectroscopy where it caused a large increase in membrane resistance. For unmodified β-CD, the extraction of cholesterol from a cholesterol containing bilayer was evident in a decrease in the bilayer resistance. For the first time, this direct approach to targeted delivery of a reagent to the proximal layer of a lipid bilayer offers the potential to build models of bidirectional signaling (inside-out vs outside-in) in cell membrane model systems.

Biaxial mechanical properties of bovine jugular venous valve leaflet tissues.

Venous valve incompetence has been implicated in diseases ranging from chronic venous insufficiency (CVI) to intracranial venous hypertension. However, while the mechanical properties of venous valve leaflet tissues are central to CVI biomechanics and mechanobiology, neither stress-strain curves nor tangent moduli have been reported. Here, equibiaxial tensile mechanical tests were conducted to assess the tangent modulus, strength and anisotropy of venous valve leaflet tissues from bovine jugular veins. Valvular tissues were stretched to 60% strain in both the circumferential and radial directions, and leaflet tissue stress-strain curves were generated for proximal and distal valves (i.e., valves closest and furthest from the right heart, respectively). Toward linking mechanical properties to leaflet microstructure and composition, Masson's trichrome and Verhoeff-Van Gieson staining and collagen assays were conducted. Results showed: (1) Proximal bovine jugular vein venous valves tended to be bicuspid (i.e., have two leaflets), while distal valves tended to be tricuspid; (2) leaflet tissues from proximal valves exhibited approximately threefold higher peak tangent moduli in the circumferential direction than in the orthogonal radial direction (i.e., proximal valve leaflet tissues were anisotropic; [Formula: see text]); (3) individual leaflets excised from the same valve apparatus appeared to exhibit different mechanical properties (i.e., intra-valve variability); and (4) leaflets from distal valves exhibited a trend of higher soluble collagen concentrations than proximal ones (i.e., inter-valve variability). To the best of the authors' knowledge, this is the first study reporting biaxial mechanical properties of venous valve leaflet tissues. These results provide a baseline for studying venous valve incompetence at the tissue level and a quantitative basis for prosthetic venous valve design.

Phase behavior of supported lipid bilayers: A systematic study by coarse-grained molecular dynamics simulations.

Solid-supported lipid bilayers are utilized by experimental scientists as models for biological membranes because of their stability. However, compared to free standing bilayers, their close proximity to the substrate may affect their phase behavior. As this is still poorly understood, and few computational studies have been performed on such systems thus far, here we present the results from a systematic study based on molecular dynamics simulations of an implicit-solvent model for solid-supported lipid bilayers with varying lipid-substrate interactions. The attractive interaction between the substrate and the lipid head groups that are closest to the substrate leads to an increased translocation of the lipids from the distal to the proximal bilayer-leaflet. This thereby leads to a transbilayer imbalance of the lipid density, with the lipid density of the proximal leaflet higher than that of the distal leaflet. Consequently, the order parameter of the proximal leaflet is found to be higher than that of the distal leaflet, the higher the strength of lipid interaction is, the stronger the effect. The proximal leaflet exhibits gel and fluid phases with an abrupt melting transition between the two phases. In contrast, below the melting temperature of the proximal leaflet, the distal leaflet is inhomogeneous with coexisting gel and fluid domains. The size of the fluid domains increases with increasing the strength of the lipid interaction. At low temperatures, the inhomogeneity of the distal leaflet is due to its reduced lipid density.

Membrane Binding and Remodelling by the COPII Coat

Dynamic remodelling of cellular membranes in conjunction with protein complex assembly and disassembly constitutes a key component of intracellular trafficking pathways. Reconstitution of selected steps outside the cell using artificial membranes facilitates the use of biophysical techniques for investigating structure and dynamics of protein-lipid assemblies. COPII-coated membrane vesicles are responsible for transporting cargo from the ER towards the Golgi apparatus in the secretory pathway. The small GTPase Sar1, which belongs to the Sar1/Arf family of GTPases and the Ras-superfamily, is an essential component in COPII-vesicle formation. Upon activation by GTP, Sar1 binds to membranes, embedding an amphipathic helix into the proximal leaflet of the bilayer. The role of GTP presence versus GTP hydrolysis in the fission of COPII vesicles is not completely clear. We study COPII vesicle formation in a bottom-up fashion on baker's yeast proteins and model membranes using cryo-electron microscopy techniques (cryo-EM), confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS). Cryo-EM shows strongly different COPII coated vesicle morphologies under GTP hydrolyzing versus non-hydrolyzing conditions. More subtle differences are observed by cryo-EM among reconstituted COPII samples under non-hydrolyzing conditions. Membrane binding of the small GTPases Sar1 and Arf1 is studied with biophysical techniques: By combining FCS with a Langmuir film balance, the protein's footprint in the proximal membrane leaflet is revealed. Dual-color fluorescence cross-correlation spectroscopy (FCCS) analysis has been developed into a method for obtaining binding curves and testing binding models of protein binding to freely diffusing vesicles. The higher specificity makes fluorescence cross-correlation advantageous compared to previous fluorescence autocorrelation analysis also for small vesicle binding studies.

Transport and Organization of Cholesterol in a Planar Solid-Supported Lipid Bilayer Depend on the Phospholipid Flip-Flop Rate

Understanding the transport behavior of the cholesterol molecules within a cell membrane is a key challenge in cell biology at present. Here, we have applied sum frequency generation vibrational spectroscopy to characterize the transport and organization of cholesterol in different kinds of planar solid-supported lipid bilayers by combining achiral- and chiral-sensitive polarization measurements. This method allows us to distinguish the organization of cholesterol in tail-to-tail, head-to-tail, head-to-head, and side-by-side manners. It is found that the movement of cholesterol in the lipid bilayer largely depends on the flip-flop rate of the phospholipid. The flip-flop dynamics of the phospholipid and cholesterol are synchronous. In the solid-supported zwitterionic phosphocholine lipid bilayer, the cholesterol molecules flip quickly from the distal leaflet to the neutral proximal leaflet of the bilayer and form tail-to-tail organization on both leaflets. The phosphocholine lipid and cholesterol show the same flip-flop rate. However, when the proximal leaflet is prepared using negative glycerol phospholipids, cholesterol organizes itself by mainly forming an α-β structure on the distal leaflet. Because of the strong interaction between the glycerol phospholipid and the substrate, no or only partial cholesterol molecules flip from the distal leaflet to the negatively charged proximal leaflet. However, the cholesterol molecules undergo flip-flop in the presence of salt solution because the ions weaken the interaction between the negative phospholipid and the substrate.

Super-resolution Stimulated Emission Depletion-Fluorescence Correlation Spectroscopy Reveals Nanoscale Membrane Reorganization Induced by Pore-Forming Proteins.

Membrane-protein interactions play a central role in membrane mediated cellular processes ranging from signaling, budding, and fusion, to transport across the cell membrane. Of particular significance is the process of efficient protein olgomerization and transmembrane pore formation on the membrane surface; the primary virulent pathway for the action of antimicrobial peptides and pore forming toxins (PFTs). The suggested nanoscopic length scales and dynamic nature of such membrane lipid-protein interactions makes their detection extremely challenging. Using a combination of super-resolution stimulated emission depletion nanoscopy with fluorescence correlation spectroscopy (STED-FCS) we unravel the emergence of nanoscale lateral heterogeneity in supported bilayer membranes made up of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol upon interaction with the PFT, listeriolysin O (LLO). A distinct length scale-dependent dynamical crossover (<200 nm) from a Brownian diffusive regime is observed at 33 and 50% cholesterol compositions, indicating the partitioning of lipids into domains with variable cholesterol content. At 25% cholesterol content, this dyamical crossover is observed only in bilayers incubated with LLO providing evidence for the existence of sub ∼100 nm dynamical lipid nanodomains bound to LLO pore assemblies. By introducing asymmetry in cholesterol composition across the bilayer leaflets we infer that this domain formation is driven largely due to active cholesterol sequestration and transient trapping of lipids to the membrane bound motifs present in the toxins, en route to LLO oligomerization and subsequent pore formation. Bilayers prepared with labeled lipids present in either the proximal or distal leaflet allow us to track the dynamical perturbation in a leaflet-dependent manner upon LLO incubation. From the differences in the extent and intensity of the dynamical crossover as observed with STED-FCS, these experiments reveal that the affinity for cholesterol in the membrane binding motifs of the LLO subdomains induce cholesterol and lipid reorganization to a greater extent in the distal (upper) leaflet when compared with the proximal (lower) leaflet. The observed length scale-dependent membrane reorganization that occurs due to invasion by LLO could be generalized to other cholesterol-dependent cytolysins and emphasizes the significant advantage of using super-resolution STED nanoscopy to unravel complex lipid-protein interactions in membrane and cellular biophysics.

Quantifying Membrane Binding of the GTPase Sar1 by Dual-Color Fluorescence Cross-Correlation Spectroscopy

COPII vesicles are responsible for transporting cargo from the ER towards the Golgi apparatus in the secretory pathway.The small GTPase Sar1, which belongs to the Ras-superfamily, is an essential component in COPII-vesicle formation. Upon activation with GTP, Sar1 binds to membranes, embedding an amphipathic helix into the proximal leaflet of the bilayer. The exact role of GTP presence versus GTP hydrolysis in the formation in COPII vesicle fission is still controversial. Moreover, a coat is still formed under GTP hydrolyzing conditions, albeit of different structure. We study COPII vesicle formation in a bottom-up fashion on S. cerivisiae proteins using cryo-EM, confocal imaging, fluorescence correlation spectroscopy (FCS) and dual-color fluorescence cross-correlation spectroscopy (FCCS). FCS on a Langmuir film balance reveals the protein's footprint, while FCCS-analysis of Sar1 binding to liposomes yields typical binding parameters. Quantitative fluorecence cross-correlation spectroscopy is enabled by a specially developed calibration standard and artifact corrections. This way, FCCS becomes a convenient tool for analyzing protein/lipid binding.

Lateral suppressor and Goblet act in hierarchical order to regulate ectopic meristem formation at the base of tomato leaflets.

In seed plants, new axes of growth are established by the formation of meristems, groups of pluripotent cells that maintain themselves and initiate the formation of lateral organs. After embryonic development, secondary shoot meristems form in the boundary zones between the shoot apical meristem and leaf primordia, the leaf axils. In addition, many plant species develop ectopic meristems at different positions of the plant body. In the compound tomato leaf, ectopic meristems can initiate at the base of leaflets, which are delimited by two distinct boundary zones, referred to as the proximal (PLB) and distal (DLB) leaflet boundaries. We demonstrate that the two leaflet boundaries differ from each other and that ectopic meristem formation is strictly limited to the DLB. Our data suggest that the DLB harbours a group of pluripotent cells that seems to be the launching pad for meristem formation. Initiation of these meristems is dependent on the activities of the transcriptional regulators Goblet (Gob) and Lateral suppressor (Ls), specifically expressed in the DLB. Gob and Ls act in hierarchical order, because Ls transcript accumulation is dependent on Gob activity, but not vice versa. Ectopic meristem formation at the DLB is also observed in other seed plants, like Cardamine pratensis, indicating that it is part of a widespread developmental program. Ectopic meristem formation leads to an increase in the number of buds, enhances the capacity for survival and opens the route to vegetative propagation.