Intermembrane Distance Research Articles

Water Hydrogen-Bond Mediated Layer by Layer Alignment of Lipid Rafts as a Precursor of Intermembrane Processes.

Lipid rafts, which are dynamic nanodomains in the plasma membrane, play a crucial role in intermembrane processes by clustering together and growing in size within the plane of the membrane while also aligning with each other across different membranes. However, the physical origin of layer by layer alignment of lipid rafts remains to be elucidated. Here, by using fluorescence imaging and synchrotron X-ray reflectivity in a phase-separated multilayer system, we find that the alignment of raft-mimicking Lo domains is regulated by the distance between bilayers. Molecular dynamics simulations reveal that the aligned state is energetically preferred when the intermembrane distance is small due to its ability to minimize the volume of surface water, which has fewer water hydrogen bonds (HBs) compared to bulk water. Our results suggest that water HB-driven alignment of lipid rafts plays a role as a precursor of intermembrane processes such as cell-cell fusion, virus entry, and signaling.

Metal-Induced Energy Transfer (MIET) Imaging of Cell Surface Engineering with Multivalent DNA Nanobrushes.

The spacing between cells has a significant impact on cell-cell interactions, which are critical to the fate and function of both individual cells and multicellular organisms. However, accurately measuring the distance between cell membranes and the variations between different membranes has proven to be a challenging task. In this study, we employ metal-induced energy transfer (MIET) imaging/spectroscopy to determine and track the intermembrane distance and variations with nanometer precision. We have developed a DNA-based molecular adhesive called the DNA nanobrush, which serves as a cellular adhesive for connecting the plasma membranes of different cells. By manipulating the number of base pairs within the DNA nanobrush, we can modify various aspects of membrane-membrane interactions such as adhesive directionality, distance, and forces. We demonstrate that such nanometer-level changes can be detected with MIET imaging/spectroscopy. Moreover, we successfully employed MIET to measure distance variations between a cellular plasma membrane and a model membrane. This experiment not only showcases the effectiveness of MIET as a powerful tool for accurately quantifying membrane-membrane interactions but also validates the potential of DNA nanobrushes as cellular adhesives. This innovative method holds significant implications for advancing the study of multicellular interactions.

Entropic pressure on fluctuating solid membranes

Biological and crystalline membranes exhibit noticeable fluctuations at room temperature due to their low bending stiffness. These fluctuations have a significant impact on their overall mechanical behavior and interactions with external objects. When two membranes come into proximity, they mutually suppress each other’s fluctuations, leading to a repulsive force that plays a pivotal role in the mechanical behavior of these membranes. From the mechanics point of view, crystalline membranes are modeled as solid membranes with inherent shear resistance, whereas biological membranes are commonly described as fluidic entities without shear resistance. Under this premise, the entropic force between two fluctuating biological membranes is proposed to scale as p∝1/d3, where d is the intermembrane distance. Yet, there are numerous instances where these membranes display shear resistance and behave akin to solid membranes. In this paper, we develop a statistical mechanics model within nonlinear elasticity to study the entropic force acting on a confined, fluctuating solid membrane. We demonstrate that, due to the nonlinear elasticity of solid membranes, the entropic force scales differently compared to that of fluid membranes. Our predictions align well with the results obtained from molecular dynamics simulations involving graphene, a representative of a solid membrane, confined between two rigid walls.

Vascular Endothelial Barrier Protection Prevents Atrial Fibrillation by Preserving Cardiac Nanostructure

BackgroundAtrial fibrillation (AF), the most common cardiac arrhythmia, is widely associated with inflammation, vascular dysfunction, and elevated levels of the vascular leak-inducing cytokine, vascular endothelial growth factor (VEGF). Mechanisms underlying AF are poorly understood and current treatments only manage this progressive disease, rather than arresting the underlying pathology. The authors previously identified edema-induced disruption of sodium channel (NaV1.5)-rich intercalated disk nanodomains as a novel mechanism for AF initiation secondary to acute inflammation. Therefore, we hypothesized that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. ObjectivesIn this study the authors tested the hypothesis that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. They identified 2 molecular targets for vascular barrier protection, connexin43 (Cx43) hemichannels and pannexin-1 (Panx1) channels, which have been implicated in cytokine-induced vascular leak. MethodsThe authors undertook in vivo electrocardiography, electron microscopy, and super-resolution light microscopy studies in mice acutely treated with a clinically relevant level of VEGF. ResultsAF incidence was increased in untreated mice exposed to VEGF relative to vehicle control subjects. VEGF also increased the average number of AF episodes. VEGF shifted NaV1.5 signal to longer distances from Cx43 gap junctions, measured by a distance transformation-based spatial analysis of 3-dimensional confocal images of intercalated disks. Similar effects were observed with NaV1.5 localized near mechanical junctions composed of neural cadherin. Blocking connexin43 hemichannels (αCT11 peptide) or Panx1 channels (PxIL2P peptide) significantly reduced the duration of AF episodes compared with VEGF alone with no treatment. Concurrently, both peptide therapies preserved NaV1.5 distance from gap junctions to control levels and reduced mechanical junction-adjacent intermembrane distance in these hearts. Notably, similar antiarrhythmic efficacy was also achieved with clinically-relevant small-molecule inhibitors of Cx43 and Panx1. ConclusionsThese results highlight vascular barrier protection as an antiarrhythmic strategy following inflammation-induced vascular leak.

Membrane Tension Inhibits Lipid Mixing by Increasing the Hemifusion Stalk Energy.

Fusion of biological membranes is fundamental in various physiological events. The fusion process involves several intermediate stages with energy barriers that are tightly dependent on the mechanical and physical properties of the system, one of which is membrane tension. As previously established, the late stages of fusion, including hemifusion diaphragm and pore expansions, are favored by membrane tension. However, a current understanding of how the energy barrier of earlier fusion stages is affected by membrane tension is lacking. Here, we apply a newly developed experimental approach combining micropipette-aspirated giant unilamellar vesicles and optically trapped membrane-coated beads, revealing that membrane tension inhibits lipid mixing. We show that lipid mixing is 6 times slower under a tension of 0.12 mN/m compared with tension-free membranes. Furthermore, using continuum elastic theory, we calculate the dependence of the hemifusion stalk formation energy on membrane tension and intermembrane distance and find the increase in the corresponding energy barrier to be 1.6 kBT in our setting, which can explain the increase in lipid mixing time delay. Finally, we show that tension can be a significant factor in the stalk energy if the pre-fusion intermembrane distance is on the order of several nanometers, while for membranes that are tightly docked, tension has a negligible effect.

Impact of the inner solute structure on the electrostatic mean-field and strong-coupling regimes of macromolecular interactions.

The structural diversity of the solute molecules involved in biomolecular processes necessitates the characterization of the forces between charged macromolecules beyond the point-ion description. From the field-theoretic partition function of an electrolyte confined between two anionic membranes, we derive a contact-value identity valid for general intramolecular solute structure and electrostatic coupling strength. In the electrostatic mean-field regime, the inner charge spread of the solute particles is shown to induce the twofold enhancement of the short-range Poisson-Boltzmann level membrane repulsion and a longer-range depletion attraction. Our contact theorem indicates that the twofold repulsion enhancement by solute size is equally present in the opposite strong-coupling regime of linear and spherical solute molecules. Upon the inclusion of the dielectric contrast between the electrolyte and the interacting membranes, the emerging polarization forces substantially amplify the solute specificity of the macromolecular interactions. Namely, the finite size of the dumbbell-like solute particles composed of similar terminal charges weakens the intermembrane repulsion. However, the extended structure of the solute molecules carrying opposite elementary charges such as ionized atoms and zwitterionic molecules enhances the membrane repulsion by several factors. We also show that these polarization forces can extend the range of the solute structure effects up to intermembrane distances exceeding the solute size by an order of magnitude. This radical alteration of the intermembrane interactions by the salt structure identifies the solute specificity as a key ingredient of the thermodynamic stability in colloidal systems.

Hinging on Success: Leveraging the Power of CAR T-Cell Therapy through In-Silico Modeling of Hinge Length and Epitope Location

Abstract Genetic engineering of T-cells using chimeric antigen receptors (CARs) has shown promise in the treatment of relapsed/refractory B-cell leukemia. However, a significant percentage of patients will eventually relapse, often due to the emergence of tumor cells expressing low levels of the target antigen. Previous research has demonstrated that the hinge domain of the CAR plays a crucial role in regulating the cytotoxic capabilities of these cells. In this study, we assessed the potential to model optimal hinge length for a given single-chain variable fragment (scFv) in relation to the location of the targeted epitope. We designed an array of hinges originating from CD8α and incorporated these hinges into CARs targeting CD19, CD22, CD33, and CD20 antigens. We found that the location of the epitope is critical in determining the optimal hinge; a narrow range of short hinges is more effective against membrane-distal epitopes while long hinges are more effective against membrane-proximal epitopes. To build an in-silico model, we used AlphaFold2 (AF2), reducing the length of the multiple sequence alignment (MSA) clusters to introduce stochasticity and increase the conformational diversity of the structural predictions for the CAR hinges and target proteins. We discovered that CARs with a predicted intermembrane distance of 15nm between the CAR and ligand were particularly effective against antigen-low leukemic cells. Our findings suggest that intermembrane distance can predict CAR responsiveness against a specific epitope, enabling the rapid optimization of CARs by adjusting hinge length. The development of this modeling strategy will facilitate the design of CARs with optimized cytotoxic potential against a wide range of novel targets. Intramural Research Program of the National Cancer Institute (Bethesda, MD)

Abstract 1787: Design of bispecific adapters for universal CAR T cell therapies of solid tumors

Abstract Recent success in the use of chimeric antigen receptor (CAR) T cell therapies to treat hematologic cancers has encouraged the exploration of CAR T cell technologies to treat solid tumors. For establishment of a productive immunologic synapse between the CAR T cell and cancer cell, multiple CARs on the T cell surface must engage multiple tumor antigens on the cancer cell surface. Whereas these binding interactions are mediated in classical CAR T cells by direct recognition of the tumor antigen by the scFv on the CAR, in the case of universal CAR T cells this recognition must be created by a bispecific adapter that can bridge between the tumor antigen and the CAR. While a diversity of bispecific adapters have been explored in the literature, the bispecific adapter used here is comprised of a low molecular weight ligand that binds avidly to the desired tumor antigen, the yellow dye fluorescein (FL) that binds tightly to an anti-fluorescein scFv on the CAR, and a polyethylene glycol (PEG) spacer that spans between the two. The length of this PEG spacer appears to be critical to ensure that 1) the bispecific adapter is long enough to mediate engagement of the universal CAR T cell with the cancer cell, and 2) the resulting intercellular distance is optimal for activating the necessary co-receptor interactions.In our study, we have utilized protein structure data and molecular modeling to estimate the optimal adapter spacer lengths for bridging between our CAR T cell and two important target cells, namely PSMA positive prostate cancer cells and fibroblast activation protein (FAP) expressing cancer associated fibroblasts. We then synthesized the desired bispecific adapters with a variety lengths of PEG spacer and evaluated their effects in mediating the cytotoxicity of our universal anti-FL CAR T cells both in vitro and in vivo. Our results reveal that the optimal spacer lengths in the bispecific adapters for targeting PSMA (DUPA-FL) and FAP (FAPL-FL) are different and depend on the locations of the adapter binding pockets on their respective protein targets, as predicted by molecular modeling. Specifically, a shorter PEG spacer is required for maximal cytoxicity when employing the PSMA-targeted adapter, whereas a much longer PEG spacer is needed to achieve maximal cell killing with the FAP-targeted adapter. In addition, spacer length can also be adjusted to avoid overactivation-induced T cell exhaustion. Taken together, our studies demonstrate that instead of optimizing the hinge length of a CAR for each antigen epitope, our bispecific adapter/universal CAR platform provides a highly flexible strategy for optimizing the intermembrane distance between the universal CAR T cell and cancer cell to achieve maximal target cell killing with minimal CAR T cell exhaustion. Citation Format: Suilan Zheng, Bo Huang, Yashapal Singh, Haiyan Chu, Suresh K. Bowroju, Md Sazzadul Bari, Madduri Srinivasarao, Sudarsan R. Kasireddy, Ramesh Mukkamala, Laurie Beitz, Byoung Ryu, Andrew M. Scharenberg, Michael Jensen, Philip S. Low. Design of bispecific adapters for universal CAR T cell therapies of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1787.

Membrane-anchored DNA nanojunctions enable closer antigen-presenting cell-T-cell contact in elevated T-cell receptor triggering.

How the engagement of a T-cell receptor to antigenic peptide-loaded major histocompatibility complex on antigen-presenting cells (APCs) initiates intracellular signalling cascades in T cells is not well understood. In particular, the dimension of the cellular contact zone is regarded as a determinant, but its influence remains controversial. This is due to the need for appropriate strategies for manipulating intermembrane spacing between the APC-T-cell interfaces without involving protein modification. Here we describe a membrane-anchored DNA nanojunction with distinct sizes to extend, maintain and shorten the APC-T-cell interface down to 10 nm. Our results suggest that the axial distance of the contact zone is critical in T-cell activation, presumably by modulating protein reorganization and mechanical force. Notably, we observe the promotion of T-cell signalling by shortening the intermembrane distance.

Quantitative similarity of HIV gp160 V513E-dominant reduction of fusion and infection with fusion by the gp41 ectodomain hairpin supports an important fusion role for the final trimer-of-hairpins structure.

The V513E mutation of the gp41 subunit of HIV gp160 has been known for thirty years to dominantly reduce HIV fusion and infection, which has been important to estimate the number of trimers needed for efficient infection. This mutation is at the N-terminus of the fusion peptide (Fp) region of gp41 that binds the target membrane. The structural basis for V513E-dominant reduction is not yet well-understood. In this study, mixtures of WT and V513E proteins of a large HIV gp41 ectodomain construct with final trimer-of-hairpins structure exhibit V513E-dominant reduction of vesicle fusion that is quantitatively-similar to that for gp160 fusion and infection. The gp41 ectodomain also exhibits V513E-dominant reduction of hairpin helicity. Global fitting of the gp41 ectodomain and gp160 data supports a requirement of six gp41/two trimers for most efficient fusion and infection. The data support a model in which the ∼25 kcal/mole free energy for close membrane apposition prior to membrane fusion is compensated by the thermostable hairpin in between Fp in target membrane and proximal and transmembrane domains in virus membrane. The Fp's are hypothesized to form an antiparallel β sheet with interleaved strands from two trimers. Relative to WT, sheets with V513E strands are longer because of salt bridges/hydrogen bonds between E2 and charged/polar residues near the Fp C-terminus. The longer V513E sheet results in shorter hairpin with consequent larger inter-membrane distance during initial apposition prior to membrane fusion. This increased distance is the hypothesized reason for reduced fusion and infection. Overall, these data support an important role for the final hairpin structure in membrane fusion.

A systematic investigation of the cardiac intercalated disk in health and disease.

During each heartbeat, action potential (AP) propagation at intercalated discs (ID), specialized structures that electrically (gap junctions [GJ]) and mechanically (adherens junctions [AJ], desmosomes [Des]) couple cardiomyocytes, coordinates the contraction of billions of cardiomyocytes. Electrogenic proteins underlying excitability (cardiac sodium channels [NaV1.5], inward-rectifier potassium channels [Kir2.1] and sodium-potassium ATPase [NKA]) enriched within ID nanodomains are emerging as vital machinery for AP propagation. ID disruption is a hallmark of arrhythmic disease, yet, IDs are omitted from physiological models due to lack of structural data. We used indirect correlative light and electron microscopy [iCLEM] to quantify ID ultrastructure and molecular organization in health and disease and link them with functional properties (conduction velocity, arrhythmia vulnerability). Additional studies in hearts treated with adhesion inhibitor peptides targeting different ID nanodomains helped bridge health with complex disease. In healthy mouse hearts, transmission electron microscopy uncovered smaller, more frequent junctions and closer intermembrane distance at peri-junctional nanodomains in atria vs. ventricles. Confocal microscopy revealed greater NaV1.5, Kir2.1, NKA expression with more preferential ID enrichment in atrial myocytes than ventricular. STORM single molecule localization microscopy, a super-resolution technique, identified chamber-specific differences in NaV1.5, Kir2.1 and NKA clustering and distribution relative to AJ, Des, GJ as well as each other. Adhesion inhibitor peptides Nadp1 (target: AJ), dadp1 (target: Des) and βadp1 (target: GJ-adjacent perinexi), but not corresponding inactive control peptides, acutely (60 minutes) disrupted their respective target nanodomains and dispersed NaV1.5 clusters from those sites. These structural impacts led to conduction slowing and reentrant arrhythmias with severity proportional to the amount of NaV1.5 present at the target site. These data parallel our previous observations in atrial fibrillation (mouse models and human patients) and highlight NaV1.5-rich ID nanodomains as potential targets for antiarrhythmic therapy.

In situ structural analysis reveals membrane shape transitions during autophagosome formation

Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (cryo-ET), and extensive data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. The analysis uncovers an unexpectedly thin intermembrane distance that is dilated at the phagophore rim. Mapping of individual autophagic structures onto a timeline based on geometric features reveals a dynamical change of membrane shape and curvature in growing phagophores. Moreover, our tomograms show the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles, such as the vacuole and the endoplasmic reticulum (ER). Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores toward closure without a templating cargo.

Mapping Activity-Dependent Quasi-stationary States of Mitochondrial Membranes with Graphene-Induced Energy Transfer Imaging.

Graphene-induced energy transfer (GIET) was recently introduced for sub-nanometric axial localization of fluorescent molecules. GIET relies on near-field energy transfer from an optically excited fluorophore to a single sheet of graphene. Recently, we demonstrated its potential by determining the distance between two leaflets of supported lipid bilayers. Here, we use GIET imaging for mapping quasi-stationary states of the inner and outer mitochondrial membranes before and during adenosine triphosphate (ATP) synthesis. We trigger the ATP synthesis state in vitro by activating mitochondria with precursor molecules. Our results demonstrate that the inner membrane approaches the outer membrane, while the outer membrane does not show any measurable change in average axial position upon activation. The inter-membrane space is reduced by ∼2 nm. This direct experimental observation of the subtle dynamics of mitochondrial membranes and the change in intermembrane distance upon activation is relevant for our understanding of mitochondrial function.

Membrane-mediated interaction of intercellular cylindrical nanoparticles

Nanoparticles in intercellular gaps, junctions, or seals could have close contact with neighboring cells simultaneously. Understanding the interaction between intercellular nanoparticles and confining cell membranes is of fundamental importance, not only to the unravelling of endocytic mechanisms but also to implications such as controlled drug delivery in tumor tissues. Here we theoretically examine the mechanical behaviors of adhesive cylindrical nanoparticles confined between two lipid membrane patches of finite size. As the size of the particle-membrane contact region or wrapping degree increases, neighboring cylindrical nanoparticles become separated and the nanoparticle distance increases first and then decreases until the particles are fully trapped by adjacent membrane patches. Depending on the nanoparticle size, adhesion energy, membrane bending rigidity and tension, and intermembrane distance, three characteristic particle-membrane interaction phases are determined as no wrapping, partial trapping, and full trapping, and the corresponding interaction phase diagram is established. Further energy comparison indicates that multiple nanoparticles undergoing the two-membrane trapping process do not exhibit cooperative effects. Analytical estimations on the system energy and configurations at equilibrium are performed based on the force balance of the membranes at small deformation and match well with numerical solutions. The results shed light on the mechanical behaviors of multiple nanoparticles in cell junctions or gaps and may have implications for drug delivery in tumor tissues.

Height, but not binding epitope, affects the potency of synthetic TCR agonists

Under physiological conditions, peptide-major histocompatibility complex (pMHC) molecules can trigger T cell receptors (TCRs) as monovalent ligands that are sparsely distributed on the plasma membrane of an antigen-presenting cell. TCRs can also be triggered by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit nonphysiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCRβ Fab′ antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab′-DNA ligand efficiently triggers TCR as a monomer when membrane associated and exhibits a potency and activation profile resembling agonist pMHC. In this report, we explore the geometric requirements for efficient TCR triggering and cellular activation by Fab′-DNA ligands. We find that T cells are insensitive to the ligand binding epitope on the TCR complex but that length of the DNA tether is important. Increasing, the intermembrane distance spanned by Fab′-DNA:TCR complexes decreases TCR triggering efficiency and T cell activation potency, consistent with the kinetic-segregation model of TCR triggering. These results establish design parameters for constructing synthetic TCR agonists that are able to activate polyclonal T cell populations, such as T cells from a human patient, in a similar manner as the native pMHC ligand.

Transitioning from electrodialysis to reverse electrodialysis stack design for energy generation from high concentration salinity gradients

In this study, stack design for high concentration gradient reverse electrodialysis operating in recycle is addressed. High concentration gradients introduce complex transport phenomena, which are exacerbated when recycling feeds; a strategy employed to improve system level energy efficiency. This unique challenge indicates that membrane properties and spacer thickness requirements may differ considerably from reverse electrodialysis for lower concentration gradients (e.g. seawater/river water), drawing closer parallels to electrodialysis stack design. Consequently, commercially available electrodialysis and reverse electrodialysis stack design was first compared for power generation from high concentration gradients. Higher gross power densities were identified for the reverse electrodialysis stack, due to the use of thinner membranes characterised by a higher permselectivity, which improved current. However, energy efficiency of the electrodialysis stack was twice that recorded for the reverse electrodialysis stack at low current densities, which was attributed to: (i) an increased residence time provided by the larger intermembrane distance, and (ii) reduced exergy losses of the electrodialysis membranes, which provided comparatively lower water permeance. Further in-depth investigation into membrane properties and spacer thickness identified that membranes characterised by an intermediate water permeability and ohmic resistance provided the highest power density and energy efficiency (Neosepta ACS/CMS), while wider intermembrane distances up to 0.3 mm improved energy efficiency. This study confirms that reverse electrodialysis stacks for high concentration gradients in recycle therefore demand design more comparable to electrodialysis stacks to drive energy efficiency, but when selecting membrane properties, the trade-off with permselectivity must also be considered to ensure economic viability.

Nodal modulator (NOMO) is required to sustain endoplasmic reticulum morphology

The endoplasmic reticulum (ER) is a membrane-bound organelle responsible for protein folding, lipid synthesis, and calcium homeostasis. Maintenance of ER structural integrity is crucial for proper function, but much remains to be learned about the molecular players involved. To identify proteins that support the structure of the ER, we performed a proteomic screen and identified nodal modulator (NOMO), a widely conserved type I transmembrane protein of unknown function, with three nearly identical orthologs specified in the human genome. We found that overexpression of NOMO1 imposes a sheet morphology on the ER, whereas depletion of NOMO1 and its orthologs causes a collapse of ER morphology concomitant with the formation of membrane-delineated holes in the ER network positive for the lysosomal marker lysosomal-associated protein 1. In addition, the levels of key players of autophagy including microtubule-associated protein light chain 3 and autophagy cargo receptor p62/sequestosome 1 strongly increase upon NOMO depletion. In vitro reconstitution of NOMO1 revealed a “beads on a string” structure likely representing consecutive immunoglobulin-like domains. Extending NOMO1 by insertion of additional immunoglobulin folds results in a correlative increase in the ER intermembrane distance. Based on these observations and a genetic epistasis analysis including the known ER-shaping proteins Atlastin2 and Climp63, we propose a role for NOMO1 in the functional network of ER-shaping proteins.

Nanoscale architecture of a VAP-A-OSBP tethering complex at membrane contact sites

Membrane contact sites (MCS) are subcellular regions where two organelles appose their membranes to exchange small molecules, including lipids. Structural information on how proteins form MCS is scarce. We designed an in vitro MCS with two membranes and a pair of tethering proteins suitable for cryo-tomography analysis. It includes VAP-A, an ER transmembrane protein interacting with a myriad of cytosolic proteins, and oxysterol-binding protein (OSBP), a lipid transfer protein that transports cholesterol from the ER to the trans Golgi network. We show that VAP-A is a highly flexible protein, allowing formation of MCS of variable intermembrane distance. The tethering part of OSBP contains a central, dimeric, and helical T-shape region. We propose that the molecular flexibility of VAP-A enables the recruitment of partners of different sizes within MCS of adjustable thickness, whereas the T geometry of the OSBP dimer facilitates the movement of the two lipid-transfer domains between membranes.

Intermembrane distances at the feto-maternal interface in epitheliochorial placentation

IntroductionIn an epitheliochorial placenta, the apical membranes of trophoblast cells and of uterine epithelial cells are in contact to each other (feto-maternal contact). In addition, there are also folds in which the trophoblast membrane is in contact with itself (feto-fetal contact) and areas where apical uterine epithelial membrane is in contact with itself (materno-maternal contact). MethodsWe use transmission electron microscopy of placental samples from pigs.(n = 3), cows (n = 2), sheep (n = 2), goat (n = 2) and roe deer (n = 1) to study the intermembrane distance in these three contact types. ResultsThe measured intermembrane distances vary between 8 and 25 nm. One common feature is that the distance at feto-fetal contact sites is about 6–10 nm wider than at materno-maternal sites and feto-maternal sites show intermediate values. DiscussionThis finding suggests that the membrane distance at feto-maternal contact sites is determined by heterophilic binding of larger fetal to smaller maternal binding molecules. Homophilic binding of smaller maternal or larger fetal molecules lead to the smaller or wider intermembrane distances at materno-maternal or feto-fetal contact sites respectively. The observation that this similar pattern of membrane distances is present in pigs and in ruminants suggest that an evolutionary mechanism is involved in determining the intermembrane distance in epitheliochorial placentas.

Mechanics of cell interaction with intercellular nanoparticles: Shape-dependent competition between two-membrane trapping and single-membrane wrapping

Understanding the mechanical behaviors of nanoparticles confined between adjacent cells is of fundamental importance to the passage of drug cargoes through cell junctions, where the nanoparticles could have contact with two cell membranes simultaneously. Here we theoretically investigate the adhesive membrane wrapping and trapping of rigid cylindrical and spherical nanoparticles confined by two cell membranes. It is shown that the system energy and configurations depend collectively on the membrane bending rigidity, tension, adhesion energy, and the ratio between the particle size and intermembrane distance. Two fundamental modes of particle–membrane interaction are revealed, two-membrane trapping and single-membrane wrapping. Confined cylindrical nanoparticles remain trapped by two cell membranes and confined spherical nanoparticles could undergo a state transition from two-membrane trapping to single-membrane wrapping as the wrapping degree increases. Corresponding interaction phase diagrams for cylindrical and spherical nanoparticles are established based on the stability of no-wrapping, partial-trapping, partial-wrapping, full-trapping and full-wrapping states. Analytical predictions are also obtained in characterizing the single-membrane wrapping at early wrapping degree. Our results suggest that cylindrical nanoparticles in comparison with spherical nanoparticles might display superior drug penetration in tumors and across epithelial and endothelial barriers, providing guidance in the rational design of anticancer nanomedicine.