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Maintenance of lipid asymmetry across the two leaflets of the plasma membrane (PM) bilayer is a ubiquitous feature of eukaryotic cells. Loss of this asymmetry has been widely associated with cell death. However, increasing evidence points to the physiological importance of non-apoptotic, transient changes in PM asymmetry. Such transient scrambling events are associated with a range of biological functions, including intercellular communication and intracellular signaling. Thus, regulation of interleaflet lipid distribution in the PM is a broadly important but underappreciated cellular process with key physiological and structural consequences. Here, we compile the mounting evidence revealing multifaceted, functional roles of PM asymmetry and transient loss thereof. We discuss the consequences of reversible asymmetry on PM structure, biophysical properties and interleaflet coupling. We argue that despite widespread recognition of broad aspects of membrane asymmetry, its importance in cell biology demands more in-depth investigation of its features, regulation, and physiological and pathological implications. The asymmetric distribution of lipids, including cholesterol, in biological membranes established actively by flippases and scramblases has structural, biophysical and functional consequences in cells and implications for communication across membranes.

Zeta potential is often used to approximate a nanoparticle’s surface charge, i.e., cationic, anionic, or neutral character, and has become a standard characterization technique to evaluate nanoparticle surfaces. While useful, zeta potential values provide only very general conclusions about surface charge character. Without a thorough understanding of the measurement parameters and limitations of the technique, these values can become meaningless. This case study attempts to explore the sensitivity of zeta potential measurement using specifically formulated cationic, anionic, and neutral liposomes. This study examines zeta potential dependence on pH and ionic strength, resolving power, and highlights the sensitivity of zeta potential to charged liposomes. Liposomes were prepared with cholesterol, 1,2-distearoyl- sn -glycero-3-phosphocholine (DSPC), and varying amounts of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-dioleoyl- sn -glycero-3-phospho- l -serine (DOPS). A strong linear relationship was noted between zeta potential values and the mole percentage of charged lipids within a liposome (e.g., cationic DOTAP or anionic DOPS). This finding could be used to formulate similar liposomes to a specific zeta potential, potentially of importance for systems sensitive to highly charged species. In addition, cationic and anionic liposomes were titrated with up to two mole percent of the neutral lipid 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[methoxy(polyethylene glycol)-2000] (lipid-PEG; LP). Very small amounts of the lipid-PEG (<0.2 mol%) were found to impart stability to the DOTAP- and DOPS-containing liposomes without significantly affecting other physicochemical properties of the formulation, providing a simple approach to making stable liposomes with cationic and anionic surface charge.

Artificial lipid-bilayer membranes are valuable tools for the study of membrane structure and dynamics. For applications such as the study of vesicular transport and drug delivery, there is a pressing need for artificial vesicles with controlled size. However, controlling vesicle size and shape with nanometre precision is challenging, and approaches to achieve this can be heavily affected by lipid composition. Here, we present a bio-inspired templating method to generate highly monodispersed sub-100-nm unilamellar vesicles, where liposome self-assembly was nucleated and confined inside rigid DNA nanotemplates. Using this method, we produce homogeneous liposomes with four distinct predefined sizes. We also show that the method can be used with a variety of lipid compositions and probe the mechanism of templated liposome formation by capturing key intermediates during membrane self-assembly. The DNA nanotemplating strategy represents a conceptually novel way to guide lipid bilayer formation and could be generalized to engineer complex membrane/protein structures with nanoscale precision. Precise control of vesicle size is highly desirable both for basic biochemical research and biomedical applications. Now, monodispersed sub-100-nm vesicles with predefined sizes have been produced using a method based on membrane self-assembly within a DNA-nanostructure guide.

Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid–lipid and lipid–membrane protein interactions involved in the regulation of cellular functions. Cell-sized asymmetric giant lipid vesicles containing a very small amount of organic solvent have now been formed via inhomogeneous break-up of a lipid microtube that was generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles were used to investigate the dynamic responses of lipid molecules and the effect of asymmetry on biochemical reactions.

The exposure of phosphatidylserine (PtdSer) to the cell surface is regulated by the down-regulation of flippases and the activation of scramblases. Xkr8 has been identified as a scramblase that is activated during apoptosis, but its exogenous expression in the mouse Ba/F3 pro B cell line induces constitutive PtdSer exposure. Here we found that this Xkr8-mediated PtdSer exposure occurred at 4 °C, but not at 20 °C, although its scramblase activity was observed at 20 °C. The Xkr8-mediated PtdSer exposure was inhibited by a kinase inhibitor and enhanced by phosphatase inhibitors. Phosphorylated Xkr8 was detected by Phos-tag PAGE, and a mass spectrometric and mutational analysis identified three phosphorylation sites. Their phosphomimic mutation rendered Xkr8 resistant to the kinase inhibitor for PtdSer exposure at 4 °C, but unlike phosphatase inhibitors, it did not induce constitutive PtdSer exposure at 20 °C. On the other hand, when the flippase genes were deleted, the Xkr8 induced constitutive PtdSer exposure at high temperature, indicating that the flippase activity normally counteracted Xkr8’s ability to expose PtdSer. These results indicate that PtdSer exposure can be increased by the phosphorylation-mediated activation of Xkr8 scramblase and flippase down-regulation.

Kallikrein (PKa), generated by activation of its precursor prekallikrein (PK), plays a role in the contact activation phase of coagulation and functions in the kallikrein-kinin system to generate bradykinin. The general dogma has been that the contribution of PKa to the coagulation cascade is dependent on its action on FXII. Recently this dogma has been challenged by studies in human plasma showing thrombin generation due to PKa activity on FIX and also by murine studies showing formation of FIXa-antithrombin complexes in FXI deficient mice. In this study, we demonstrate high-affinity binding interactions between PK(a) and FIX(a) using surface plasmon resonance and show that these interactions are likely to occur under physiological conditions. Furthermore, we directly demonstrate dose- and time-dependent cleavage of FIX by PKa in a purified system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and chromogenic assays. By using normal pooled plasma and a range of coagulation factor-deficient plasmas, we show that this action of PKa on FIX not only results in thrombin generation, but also promotes fibrin formation in the absence of FXII or FXI. Comparison of the kinetics of either FXIa-or PKa-induced activation of FIX suggest that PKa could be a significant physiological activator of FIX. Our data indicate that the coagulation cascade needs to be redefined to indicate that PKa can directly activate FIX. The circumstances that drive PKa substrate specificity remain to be determined.

SignificanceIn healthy cells, phosphatidylserine (PtdSer) is exclusively localized at inner leaflets of plasma membranes. When cells undergo apoptosis, caspases cleave a membrane protein, XK-related protein (Xkr) 8, at the C terminus, and the cleaved Xkr8 supports scrambling of phospholipids between inner and outer leaflets of plasma membranes. The PtdSer, thus exposed to the cell surface, is recognized by macrophages as an “eat me” signal for engulfment of dead cells. We report that Xkr8 is present as a complex with basigin or neuroplastin that work as a chaperone to localize Xkr8 at plasma membranes. When Xkr8 is cleaved by caspase, the complex undergoes a higher-order structure. These results will contribute to our understanding of how the Xkr8 scrambles phospholipids when cells undergo apoptosis. Xk-related protein (Xkr) 8, a protein carrying 10 transmembrane regions, is essential for scrambling phospholipids during apoptosis. Here, we found Xkr8 as a complex with basigin (BSG) or neuroplastin (NPTN), type I membrane proteins in the Ig superfamily. In BSG−/−NPTN−/− cells, Xkr8 localized intracellularly, and the apoptosis stimuli failed to expose phosphatidylserine, indicating that BSG and NPTN chaperone Xkr8 to the plasma membrane to execute its scrambling activity. Mutational analyses of BSG showed that the atypical glutamic acid in the transmembrane region is required for BSG’s association with Xkr8. In cells exposed to apoptotic signals, Xkr8 was cleaved at the C terminus and the Xkr8/BSG complex formed a higher-order complex, likely to be a heterotetramer consisting of two molecules of Xkr8 and two molecules of BSG or NPTN, suggesting that this cleavage causes the formation of a larger complex of Xkr8-BSG/NPTN for phospholipid scrambling.

Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins 1 – 3 . The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour 3 , 4 . The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that senses and responds to mechanical stress by flattening and releasing sequestered factors 5 – 9 . Here we isolated near-native eisosomes as helical tubules made up of a lattice of Pil1/Lsp1 bound to plasma membrane lipids, and solved their structures by helical reconstruction. Our structures reveal a striking organization of membrane lipids, and, using in vitro reconstitutions and molecular dynamics simulations, we confirmed the positioning of individual PI(4,5)P 2 , phosphatidylserine and sterol molecules sequestered beneath the Pil1/Lsp1 coat. Three-dimensional variability analysis of the native-source eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects the sequestration of these lipids. Collectively, our results support a mechanism in which stretching of the Pil1/Lsp1 lattice liberates lipids that would otherwise be anchored by the Pil1/Lsp1 coat, and thus provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain. Cryo-electron microscopy, in vitro reconstitution and molecular dynamics simulations provide insight into the architecture of a plasma membrane microdomain in yeast, the organization and dynamics of the membrane lipids within this microdomain and how it responds to mechanical stress.

Significance Extracellular vesicle (EV)-mediated transfer of macromolecules may play a key role in cellular communication and may have utility in directed molecular therapies. In addition, the EV packaged biomolecules in serum may have potential for diagnosing cancer and determining its likelihood of metastasis. EVs are heterogeneous and there are many outstanding questions associated with biogenesis, uptake, and the fate of transferred molecules in recipient cells. In fact, the function, characterization, and even the nomenclature of EVs are being refined. Here we aimed to improve the functional characterization of EVs, and observed that only microvesicles (MVs), but not exosomes, can functionally transfer loaded reporter molecules to recipient cells, largely by delivering plasmid DNA. Our data show that exosomes and MVs are structurally and functionally distinct. Extracellular vesicles (EVs), specifically exosomes and microvesicles (MVs), are presumed to play key roles in cell–cell communication via transfer of biomolecules between cells. The biogenesis of these two types of EVs differs as they originate from either the endosomal (exosomes) or plasma (MVs) membranes. To elucidate the primary means through which EVs mediate intercellular communication, we characterized their ability to encapsulate and deliver different types of macromolecules from transiently transfected cells. Both EV types encapsulated reporter proteins and mRNA but only MVs transferred the reporter function to recipient cells. De novo reporter protein expression in recipient cells resulted only from plasmid DNA (pDNA) after delivery via MVs. Reporter mRNA was delivered to recipient cells by both EV types, but was rapidly degraded without being translated. MVs also mediated delivery of functional pDNA encoding Cre recombinase in vivo to tissues in transgenic Cre-lox reporter mice. Within the parameters of this study, MVs delivered functional pDNA, but not RNA, whereas exosomes from the same source did not deliver functional nucleic acids. These results have significant implications for understanding the role of EVs in cellular communication and for development of EVs as delivery tools. Moreover, studies using EVs from transiently transfected cells may be confounded by a predominance of pDNA transfer.

Aggregation of human α-synuclein (αSyn) is linked to Parkinson’s disease (PD) pathology. The central region of the αSyn sequence contains the non-amyloid β-component (NAC) crucial for aggregation. However, how NAC flanking regions modulate αSyn aggregation remains unclear. Using bioinformatics, mutation and NMR, we identify a 7-residue sequence, named P1 (residues 36–42), that controls αSyn aggregation. Deletion or substitution of this ‘master controller’ prevents aggregation at pH 7.5 in vitro. At lower pH, P1 synergises with a sequence containing the preNAC region (P2, residues 45–57) to prevent aggregation. Deleting P1 (ΔP1) or both P1 and P2 (ΔΔ) also prevents age-dependent αSyn aggregation and toxicity in C. elegans models and prevents αSyn-mediated vesicle fusion by altering the conformational properties of the protein when lipid bound. The results highlight the importance of a master-controller sequence motif that controls both αSyn aggregation and function—a region that could be targeted to prevent aggregation in disease.Using computational, spectroscopic and in vivo approaches, two short motifs in the N-terminal region of human α-synuclein are shown to be critical for toxic protein aggregation but also for membrane fusion.