Freeze-fracture Electron Microscopy Research Articles

Time Dependence of Gel Formation in Lyotropic Nematic Liquid Crystals: From Hours to Weeks.

The combination of lyotropic liquid crystals (LLCs) and low-molecular-weight gelators (LMWGs) for the formation of lyotropic liquid crystal gels (LLC gels) leads to a versatile and complex material combining properties of both parent systems. We gelled the calamitic nematic NC phases of a binary and ternary system using the LMWG 3,5-bis-(5-hexylcarbamoyl-pentoxy)-benzoic acid hexyl ester (BHPB-6). This binary system consists of the surfactant N,N-dimethyl-N-ethyl-1-hexadecylammonium bromide (CDEAB) and water, whereas the ternary system consists of the surfactant N,N,N-trimethyl-N-tetradecylammonium bromide (C14TAB), the cosurfactant n-decanol, and water. Though containing similar surfactants, the gelled NC phases of the binary and ternary systems show differences in their visual and gel properties. The gelled NC phase of the binary system remains clear for several days after preparation, whereas the gelled NC phase of the ternary system turns turbid within 24 h. We investigated the time evolution of the gel strength with oscillation rheology measurements (a) within the first 24 h and (b) up to two weeks after gel formation. The shape of the fibers was investigated over different time scales with freeze fracture electron microscopy (FFEM). We demonstrate that despite their similarities, the two LLC gels also have distinct differences.

Coordinated regulation of phosphatidylinositol 4-phosphate and phosphatidylserine levels by Osh4p and Osh5p is an essential regulatory mechanism in autophagy

Macroautophagy (hereafter autophagy) is an intracellular degradative pathway in budding yeast cells. Certain lipid types play essential roles in autophagy; yet the precise mechanisms regulating lipid composition during autophagy remain unknown. Here, we explored the role of the Osh family proteins in the modulating lipid composition during autophagy in budding yeast. Our results showed that osh1-osh7∆ deletions lead to autophagic dysfunction, with impaired GFP-Atg8 processing and the absence of autophagosomes and autophagic bodies in the cytosol and vacuole, respectively. Freeze-fracture electron microscopy (EM) revealed elevated phosphatidylinositol 4-phosphate (PtdIns(4)P) levels in cytoplasmic and luminal leaflets of autophagic bodies and vacuolar membranes in all deletion mutants. Phosphatidylserine (PtdSer) levels were significantly decreased in the autophagic bodies and vacuolar membranes in osh4∆ and osh5∆ mutants, whereas no significant changes were observed in other osh deletion mutants. Furthermore, we identified defects in autophagic processes in the osh4∆ and osh5∆ mutants, including rare autophagosome formation in the osh5∆ mutant and accumulation of autophagic bodies in the vacuole in the osh4∆ mutant, even in the absence of the proteinase inhibitor PMSF. These findings suggest that Osh4p and Osh5p play crucial roles in the transport of PtdSer to autophagic bodies and autophagosome membranes, respectively. The precise control of lipid composition in the membranes of autophagosomes and autophagic bodies by Osh4p and Osh5p represents an important regulatory mechanism in autophagy.

Thylakoid membrane stacking controls electron transport mode during the dark-to-light transition by adjusting the distances between PSI and PSII.

The balance between linear electron transport (LET) and cyclic electron transport (CET) plays an essential role in plant adaptation and protection against photo-induced damage. This balance is largely maintained by phosphorylation-driven alterations in the PSII-LHCII assembly and thylakoid membrane stacking. During the dark-to-light transition, plants shift this balance from CET, which prevails to prevent overreduction of the electron transport chain and consequent photo-induced damage, towards LET, which enables efficient CO2 assimilation and biomass production. Using freeze-fracture cryo-scanning electron microscopy and transmission electron microscopy of Arabidopsis leaves, we reveal unique membrane regions possessing characteristics of both stacked and unstacked regions of the thylakoid network that form during this transition. A notable consequence of the morphological attributes of these regions, which we refer to as 'stacked thylakoid doublets', is an overall increase in the proximity and connectivity of the two photosystems (PSI and PSII) that drive LET. This, in turn, reduces diffusion distances and barriers for the mobile carriers that transfer electrons between the two PSs, thereby maximizing LET and optimizing the plant's ability to utilize light energy. The mechanics described here for the shift between CET and LET during the dark-to-light transition are probably also used during chromatic adaptation mediated by state transitions.

Cinnamic Acid and Caffeic Acid Effects on Gastric Tight Junction Proteins Analyzed in Xenopus laevis Oocytes.

Analysis of secondary plant compounds for the development of novel therapies is a common focus of experimental biomedicine. Currently, multiple health-supporting properties of plant-derived molecules are known but still information on many mechanisms is scarce. Cinnamic acid and caffeic acid are two of the most abundant polyphenols in human dietary fruits and vegetables. In this study, we investigated cinnamic acid and caffeic acid effects on the gastric barrier, which is primarily provided by members of the transmembrane tight junction protein family of claudins. The Xenopus laevis oocyte has been established, in recent years, as a heterologous expression system for analysis of transmembrane tight junction protein interactions, by performing paired oocyte experiments to identify an effect on protein-protein interactions, in vitro. In our current study, human gastric claudin-4, -5, and -18.2. were expressed and detected in the oocyte plasma membrane by freeze fracture electron microscopy and immunoblotting. Oocytes were paired and incubated with 100 µM or 200 µM cinnamic acid or caffeic acid, or Ringer's solution, respectively. Caffeic acid showed no effect on the contact area strength of paired oocytes but led to an increased contact area size. In contrast, cinnamic acid-incubated paired oocytes revealed a reduced contact area and a strengthening effect on the contact area was identified. These results may indicate that caffeic acid and cinnamic acid both show an effect on gastric barrier integrity via direct effects on tight junction proteins.

Bimodal DNA self-origami material with nucleic acid function enhancement

BackgroundThe design of DNA materials with specific nanostructures for biomedical tissue engineering applications remains a challenge. High-dimensional DNA nanomaterials are difficult to prepare and are unstable; moreover, their synthesis relies on heavy metal ions. Herein, we developed a bimodal DNA self-origami material with good biocompatibility and differing functions using a simple synthesis method. We simulated and characterized this material using a combination of oxDNA, freeze–fracture electron microscopy, and atomic force microscopy. Subsequently, we optimized the synthesis procedure to fix the morphology of this material.ResultsUsing molecular dynamics simulation, we found that the bimodal DNA self-origami material exhibited properties of spontaneous stretching and curling and could be fixed in a single morphology via synthesis control. The application of different functional nucleic acids enabled the achievement of various biological functions, and the performance of functional nucleic acids was significantly enhanced in the material. Consequently, leveraging the various functional nucleic acids enhanced by this material will facilitate the attainment of diverse biological functions.ConclusionThe developed design can comprehensively reveal the morphology and dynamics of DNA materials. We thus report a novel strategy for the construction of high-dimensional DNA materials and the application of functional nucleic acid–enhancing materials.Graphical

Multiscale organisation of lead carboxylates in artistic oil binders.

The supramolecular and mesoscopic architectures of lead-saponified linseed oil, used by painters since the Renaissance, have been characterised and linked to their rheological properties. The multi-scale organization of saponified oils has been demonstrated by SAXS (Small Angle X-ray Scattering), FF-TEM (Freeze-Fracture Transmission Electron Microscopy) and DIC (Differential Interference Contrast): some of the lead soaps (formed when the oil is heated in the presence of PbO) are organized into microscopic lamellar domains, distributed in a continuous matrix made up of unorganized species (partially saponified triglycerides, glycerol, remaining soaps, etc.). The concentration of lead soaps in the oil controls the average size and interaction between the lamellar domains. Linseed oil + PbO 17 mol% is viscous and consists of aggregates of lamellar domains isolated within the continuous unorganized matrix. In contrast, in linseed oil + PbO 50 mol%, the domains are homogeneously dispersed and form what can be described as a three-dimensional network, giving the system viscoelastic properties.

Transition from a foam-like to an onion-like nanostructure in water-rich L3 phases

Abstract In dilute water-surfactant systems L3 phases are found in which bilayers interconnect to form a sample-spanning sponge-like structure. From our previous study of the system water/NaCl-AOT (sodium bis(2-ethylhexyl) sulfosuccinate) we know that a transition of this sponge-like structure to an oil-continuous foam-like structure occurs upon addition of minute amounts of oil (about 3 wt%, α = 0.03) in the L3 channel at a constant surfactant mass fraction of γ = 0.15 and T = 25 °C. The aim of the present study was to verify if the same transition occurs at γ = 0.25. To achieve this goal, we determined the relevant part of the phase diagram and studied the electrical conductivities and viscosities within the narrow one-phase L3 channel. Although the electrical conductivities and viscosities change qualitatively like those observed at γ = 0.15 we did not observe a sponge-like structure at γ = 0.25 in the oil-free system (α = 0) with freeze-fracture electron microscopy (FFEM) and freeze fracture direct imaging (FFDI). Together with the FFEM/FFDI images and SANS/SAXS curves we provide experimental evidence for a structural transition with decreasing oil content from a thermodynamically stable foam-like to a thermodynamically stable onion-like nanostructure at γ = 0.25 rather than to a sponge-like structure as is the case at γ = 0.15.

Transition from a Sponge-Like to an Onion-Like Nanostructure in the L3 Phase – Part I

HypothesisIn the phase diagram of the system H2O/NaCl – AOT (sodium bis(2-ethylhexyl) sulfosuccinate) a homogeneous L3 channel can be detected at T = 25 °C as a function of the surfactant and the NaCl mass fraction. Our hypothesis is that a structural transition from a sponge-like to an onion-like structure occurs in this channel. ExperimentsWe (a) determined the relevant part of the phase diagram, (b) carried out electrical conductivity and viscosity measurements, (c) visualized the nanostructure with freeze-fracture electron microscopy (FFEM) and freeze fracture direct imaging (FFDI), (d) determined the characteristic length scales from Small Angle Neutron Scattering (SANS) curves. FindingsWe detected a narrow one-phase L3 channel in which the conductivities decrease, while the viscosities increase with increasing surfactant and salt mass fractions. Together with the FFEM/FFDI images and the SANS curves we provide experimental evidence for a structural transition from a sponge-like to an onion-like structure: all structures as well as their coexistences are thermodynamically stable.

Subcellular distribution of membrane lipids revealed by freeze-fracture electron microscopy.

Cell membranes are composed of a large variety of lipids and proteins. While the localization and function of membrane proteins have been extensively investigated, the distribution of membrane lipids, especially in the non-cytoplasmic leaflet of organelle membranes, remains largely unknown. Fluorescent biosensors have been widely used to study membrane lipid distribution; however, they have some limitations. By utilizing the quick-freezing and freeze-fracture replica labeling electron microscopy technique, we can uncover the precise distribution of membrane lipids within cells and assess the function of lipid-transporting proteins. In this review, I summarize recent progress in analyzing intracellular lipid distribution by utilizing this method.

Whole animal freeze-fracture scanning electron microscopy: an easy-to-use method to investigate cell type morphology of marine embryos and larvae

Morphological and molecular characterization of cell types, organs and individual organisms is essential for understanding the origins of morphogenesis. The increased implementation of high throughput methods as a means to address cell type evolution, during the last decade, created the need for an efficient way to assess cell type morphology. Here in order to create a new tool to study cell type morphology, we optimized a fast and easy-to-use whole animal freeze-fracture scanning electron microscopy (WAFFSEM) method. This method was applied on marine experimental systems (echinoderms, mollusks, tunicates, and cephalochordates), that have been widely used to assess environmental, developmental, and evolutionary questions. Our protocol does not require any specialized equipment and the processed specimens are compatible with scanning electron microscopy. This protocol was able to successfully expose the internal cell types of all specimens in which it was tested and to reveal their cellular and subcellular characteristics. We strongly believe that the combination of our protocol with other methods (e.g., light microscopy and single cell transcriptomics) will be beneficial to further improve the way to classify and describe cell types.

Gelled lyotropic nematic liquid crystals

ABSTRACT Lyotropic liquid crystal gels (LLC gels) are versatile materials which combine the anisotropy of a liquid crystal with the mechanical stability of a gel. We succeeded in gelling the rare calamitic nematic NC phase of two different micellar LLC systems with the low molecular weight gelator (LMWG) 3,5-bis-(5-hexylcarbamoyl-pentoxy)-benzoic acid hexyl ester (BHPB-6). The systems of choice are: (1) a binary LLC system with the surfactant N,N-dimethyl-N-ethyl-1-hexadecylammonium bromide (CDEAB) and water; (2) a ternary LLC system with the surfactant N,N,N-trimethyl-N-tetradecylammonium bromide (C14TAB), n-decanol, and water. The obtained gels show optical birefringence and no macroscopic flow. Freeze fracture electron microscopy (FFEM) images show ribbon-like gel fibres that form the gel network and can stack into thicker bundles, while small-angle X-ray scattering (SAXS) confirms the coexistence of the nematic NC phase and the gel network. The growth of the gel network was monitored with time-resolved SAXS measurements. Additionally, significant gel ageing effects were observed in rheology measurements, showing that the gel strengthens with time. With this study, we considerably broaden the pool of available surfactant-based LLC gels and pave a way to macroscopically aligned LLC gels with the goal to apply them as stimuli-responsive actuators and sensors.

Tight-junction strand networks and tightness of the epithelial barrier.

Tight junctions (TJs) are cell-cell junction structures critical for controlling paracellular permeability. On freeze-fracture replica electron microscopy, they appear as a continuous network of fibrils (TJ strands). TJ strands function as zippers that create a physical barrier against paracellular diffusion of molecules. The morphology of the TJ strand network varies greatly between tissues, and in recent years, studies have highlighted the mechanisms regulating the morphology of TJ strand networks and on their relevance to barrier function. In this review, we discuss evidence regarding the components of the TJ strand and the mechanisms for creating the TJ strand network. Furthermore, we discuss and hypothesize how its morphology contributes to the establishment of the epithelial barrier.

Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system

In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations—dolines—capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli.

Self-Assembled Structure of α-Isostearyl Glyceryl Ether Affects Skin Permeability-a Lamellar with 70-nm Spaces and L3 Phase Enhanced the Transdermal Delivery of a Hydrophilic Model Drug.

Self-assembled surfactant structures, such as liquid crystals, have the potential to enhance transdermal drug delivery. In the present study, the pseudo-ternary system of GET (composed of α-Isostearyl glyceryl ether (GEIS) and polysorbate 60)/1,3 butanediol (BG)/water) was shown to exhibit a complex phase diagram. Small- and wide-angle X-ray scattering (SWAXS) and freeze-fracture transmission electron microscopy (FF-TEM) revealed that GET6BG60 (6%GET/60%BG/34%Water) formed a lamellar phase with a repeated distance of approximately 72nm. Such a long-repeated distance of the lamellar phase was unique in the surfactant system. Moreover, the various structures, such as multilamellar vesicles and branched-like layers, were observed, which suggested that they might be deformable. On the other hand, only core-shell particles were observed in GET6BG20, the core of which was an L3 phase. GET6BG20 and GET6BG60 significantly enhanced the skin permeation of the hydrophilic model drug, antipyrine (ANP) (log Ko/w, - 1.51). However, their permeation profiles were distinct. Liquid chromatography-tandem mass spectrometry revealed that epidermal accumulation of GEIS was significantly higher with GET6BG60 than GET6BG20 after 1.5h of permeation, which might be attributed to differences in their deformable properties. Furthermore, GEIS was reported to affect intercellular lipids. Accumulated GEIS in the epidermis may have interacted with intercellular lipids and enhanced the transdermal delivery of ANP. The difference in the permeation profiles of ANP may be attributed to the penetration process of GEIS in the epidermis. This study suggests that GET6BG20 and GET6BG60 are unique carriers to enhance the permeation of hydrophilic drugs, such as ANP.

Molecular mechanism of claudin-15 strand flexibility: A computational study.

Claudins are one of the major components of tight junctions that play a key role in the formation and maintenance of the epithelial barrier function. Tight junction strands are dynamic and capable of adapting their structure in response to large-scale tissue rearrangement and cellular movement. Here, we present molecular dynamics simulations of claudin-15 strands of up to 225 nm in length in two parallel lipid membranes and characterize their mechanical properties. The persistence length of claudin-15 strands is comparable with those obtained from analyses of freeze-fracture electron microscopy. Our results indicate that lateral flexibility of claudin strands is due to an interplay of three sets of interfacial interaction networks between two antiparallel double rows of claudins in the membranes. In this model, claudins are assembled into interlocking tetrameric ion channels along the strand that slide with respect to each other as the strands curve over submicrometer-length scales. These results suggest a novel molecular mechanism underlying claudin-15 strand flexibility. It also sheds light on intermolecular interactions and their role in maintaining epithelial barrier function.

Freeze-Fracture Electron Microscopy for Extracellular Vesicle Analysis.

Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular communication. EVs consist of three populations of vesicles, namely microvesicles (MVs), exosomes, and apoptotic bodies. The limiting membrane of EVs is crucially involved in the interactions with the recipient cells, which could lead to the transfer of biologically active molecules to the recipient cells and, consequently, affect their behavior. The freeze-fracture electron microscopy technique is used to study the internal organization of biological membranes. Here, we present a protocol for MV isolation from cultured cancerous urothelial cells and the freeze-fracture of MVs in the steps of rapid freezing, fracturing, making and cleaning the replicas, and analyzing them with transmission electron microscopy. The results show that the protocol for isolation yields a homogenous population of EVs, which correspond to the shape and size of MVs. Intramembrane particles are found mainly in the protoplasmic face of the limiting membrane. Hence, freeze-fracture is the technique of choice to characterize the MVs' diameter, shape, and distribution of membrane proteins. The presented protocol is applicable to other populations of EVs.

Tight junction formation by a claudin mutant lacking the COOH-terminal PDZ domain-binding motif.

Claudin-based tight junctions (TJs) are formed at the most apical part of cell-cell contacts in epithelial cells. Previous studies suggest that scaffolding proteins ZO-1 and ZO-2 (ZO proteins) determine the location of TJs by interacting with claudins, but this idea is not conclusive. To address the role of the ZO proteins binding to claudins at TJs, a COOH-terminal PDZ domain binding motif-deleted claudin-3 mutant, which lacks the ZO protein binding, was stably expressed in claudin-deficient MDCK cells. The COOH-terminus-deleted claudin-3 was localized at the apicolateral region similar to full-length claudin-3. Consistently, freeze-fracture electron microscopy revealed that the COOH-terminus-deleted claudin-3-expressing cells reconstituted belts of TJs at the most apical region of the lateral membrane and restored functional epithelial barriers. These results suggest that the interaction of claudins with ZO proteins is not a prerequisite for TJ formation at the most apical part of cell-cell contacts.

(Invited, Digital Presentation) Donor-Acceptor-Linked Molecules for Controlling the Membrane Potential of Living Cells

Living animal cells have the plasma membrane potential (V m), which results from a difference in the electrical charge on the two extreme sides of the plasma membrane, that is, a slight excess of positive and negative ions outside and inside a cell, respectively. Such a difference is controlled by various ion channels. In neuronal cells, the V m is negative in the resting state and is drastically changed for neurotransmission. In response to some input, the ion channels of neuronal cells get quickly and transiently activated such that the abovementioned difference is cancelled (∆V m > 0, depolarization). Coordinated activation and deactivation of the ion channels occurs along the neuronal axons, which generates electrical pulses. This is the mechanism of neurotransmission. Therefore, the control of the V m using a light source is an attractive strategy that can allow targeted, fast control of neuronal activities. In this symposium, we have demonstrated a novel general strategy for the control of the V m by using donor-acceptor-linked molecules (D-A molecules) capable of intramolecular long-lived charge separation, nanocarriers for membrane targeting of D-A molecules, and light.The D-A molecules used in this study were triadic complexes of ferrocene (Fc), porphyrin (H2P), and fullerene (C60) (Fc-H2P-C60). We had previously reported that, under light irradiation, Fc-H2P-C60 yields the ferrocenium cation (Fc+)-H2P-C60 radical anion (C60 •-) with a long lifetime of ~0.01 ms [1]. Fc-H2P-C60 was solubilized in the physiological media with a genetically engineered lipoprotein nanoparticle (cpHDL). cpHDL was capable of high plasma membrane binding without significant cytotoxicity [2]. The Fc-H2P-C60/cpHDL complex was added to a cell culture medium of neuronal cells (rat pheochromocytoma PC12), and its plasma membrane binding was confirmed by confocal analysis and freeze-fracture electron microscopy. Subsequent light irradiation of PC12 cells decreased the membrane potential (∆V m > 0, depolarization). Such depolarization was not observed for the reference molecule (H2P alone) that was incapable of charge separation, clearly demonstrating that the charge separation was responsible for this depolarization [3]. Our following study with other D-A molecules revealed that the degree of the depolarization was positively correlated with the charge separation yield [4].To gain insight into the mechanism of this photo-induced depolarization, cytotoxicity, channel activities, and membrane capacitance were evaluated in D-A molecule-treated PC12 cells under or after irradiation. Pore formation in the plasma membrane, resulting from severe membrane damage, were not detected. Indeed, the photo-generation of reactive oxygen species (ROS) leading to membrane disruption was less efficient in the D-A molecules than in H2P incapable of photo-induced charge separation. In contrast, it was found that a specific subtype of cation channels was inhibited, and that the membrane capacitance was decreased by irradiated D-A molecules [5]. In this symposium, I will discuss more about the potential of applying our D-A molecules/carriers system in the biomedical fields.REFERENCES[1] Imahori, H. et al., J. Am. Chem. Soc. 2001, 123, 2607-2617.[2] Nakatsuji, H. et al., Angew. Chem. Int. Ed. 2015, 54, 11725-11729.[3] Numata, T. et al., J. Am. Chem. Soc. 2012, 134, 6092-6095.[4] Takano, Y. et al., Chem. Sci. 2016, 7, 3331-3337.[5] Numata, T. et al., Cell. Physiol. Biochem. 2020, 54, 899-916.

The distal C terminus of the dihydropyridine receptor β1a subunit is essential for tetrad formation in skeletal muscle

The skeletal muscle dihydropyridine receptor (DHPR) β1a subunit is indispensable for full trafficking of DHPRs into triadic junctions (i.e., the close apposition of transverse tubules and sarcoplasmic reticulum [SR]), facilitation of DHPRα1S voltage sensing, and arrangement of DHPRs into tetrads as a consequence of their interaction with ryanodine receptor (RyR1) homotetramers. These three features are obligatory for skeletal muscle excitation–contraction (EC) coupling. Previously, we showed that all four vertebrate β isoforms (β1–β4) facilitate α1S triad targeting and, except for β3, fully enable DHPRα1S voltage sensing [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. Consequently, β3 failed to restore EC coupling despite the fact that both β3 and β1a restore tetrads. Thus, all β-subunits are able to restore triad targeting, but only β1a restores both tetrads and proper DHPR–RyR1 coupling [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. To investigate the molecular region(s) of β1a responsible for the tetradic arrangement of DHPRs and thus DHPR–RyR1 coupling, we expressed loss- and gain-of-function chimeras between β1a and β4, with systematically swapped domains in zebrafish strain relaxed (β1-null) for patch clamp, cytoplasmic Ca2+ transients, motility, and freeze-fracture electron microscopy. β1a/β4 chimeras with either N terminus, SH3, HOOK, or GK domain derived from β4 showed complete restoration of SR Ca2+ release. However, chimera β1a/β4(C) with β4 C terminus produced significantly reduced cytoplasmic Ca2+ transients. Conversely, gain-of-function chimera β4/β1a(C) with β1a C terminus completely restored cytoplasmic Ca2+ transients, DHPR tetrads, and motility. Furthermore, we found that the nonconserved, distal C terminus of β1a plays a pivotal role in reconstitution of DHPR tetrads and thus allosteric DHPR–RyR1 interaction, essential for skeletal muscle EC coupling.

Stabilization of all-aqueous droplets by interfacial self-assembly of fatty acids bilayers

All-aqueous microdroplets produced by liquid–liquid phase separation have emerged as promising models of artificial cells, and offer new approaches for the solvent-free encapsulation of fragile solutes. Yet, the lack of a membrane on such droplets makes them intrinsically unstable against coarsening, and precludes a fine control over chemical localization, as solutes can freely diffuse through the interface. Herein, we report the construction of stable and impermeable water-in-water emulsions via the interfacial self-assembly of mixed sodium oleate/1-decanol bilayers on dextran-rich droplets produced by segregative liquid–liquid phase separation with poly(ethylene glycol). Lipids spontaneously self-assemble as multilamellar structures at the surface of the droplets as revealed by freeze-fracture transmission electron microscopy and small-angle X-ray scattering. We further demonstrate that the lipid-based membrane is impermeable to oligonucleotides and proteins, but also to a low molecular weight dye, so that a strict chemical encapsulation can be achieved by spontaneous partitioning within the droplets before membrane self-assembly. Taken together, our results highlight the ease of production of fatty acid-stabilized all-aqueous emulsions droplets able to encapsulate a range of solutes without the need of oil or organic solvents, paving the way to the construction of robust membrane-bounded, polymer-rich artificial cells.