We have used a modified, dual pipette assay to quantify the strength of cadherin-dependent cell–cell adhesion. The force required to separate E-cadherin–expressing paired cells in suspension was measured as an index of intercellular adhesion. Separation force depended on the homophilic interaction of functional cadherins at the cell surface, increasing with the duration of contact and with cadherin levels. Severing the link between cadherin and the actin cytoskeleton or disrupting actin polymerization did not affect initiation of cadherin-mediated adhesion, but prevented it from developing and becoming stronger over time. Rac and Cdc42, the Rho-like small GTPases, were activated when E-cadherin–expressing cells formed aggregates in suspension. Overproduction of the dominant negative form of Rac or Cdc42 permitted initial E-cadherin–based adhesion but affected its later development; the dominant active forms prevented cell adhesion outright. Our findings highlight the crucial roles played by Rac, Cdc42, and actin cytoskeleton dynamics in the development and regulation of strong cell adhesion, defined in terms of mechanical forces.

Acute metabolic changes of plasma membrane (PM) lipids, such as those mediating signaling reactions, are rapidly compensated by homeostatic responses whose molecular basis is poorly understood. Here we show that the Extended-Synaptotagmins (E-Syts), ER proteins which function as PI(4,5)P2 and Ca2+-regulated tethers to the PM, participate in these responses. E-Syts transfer glycerolipids between bilayers in vitro and such transfer requires Ca2+ and their SMP domain, a lipid-harboring module. Genome edited cells lacking E-Syts do not exhibit abnormalities in the major glycerolipids at rest, but display enhanced and sustained accumulation of PM diacylglycerol (DAG) upon PI(4,5)P2 hydrolysis by PLC activation, which can be rescued by expression of E-Syt1, but not by mutant E-Syt1 lacking the SMP domain. The formation of E-Syts-dependent ER-PM tethers in response to stimuli that cleave PI(4,5)P2 and elevate Ca2+ may help reverse accumulation of DAG in the PM by transferring it to the ER for metabolic recycling.

Neurotransmitters are released thru nascent fusion pores, which ordinarily dilate after bilayer fusion, preventing consistent biochemical studies. Here, we employ lipid bilayer nanodiscs as fusion partners, whose rigid protein framework prevents dilation and reveals properties of the SNARE-induced fusion pore. While only one SNARE per nanodisc is required for maximum rates of bilayer fusion, efficient release of content on the physiologically-relevant time scale of synaptic transmission requires ~3 or more SNAREpins and the native VAMP2 transmembrane domain. We suggest that several SNAREpins simultaneously zippering their SNARE transmembrane helices within the freshly fused bilayers provide a radial force that prevents the nascent pore from resealing during synchronous neurotransmitter release.

SummaryComplexin prevents SNAREs from releasing neurotransmitters until an action potential arrives at the synapse. To understand the mechanism for this inhibition, we determined the structure of complexin bound to a mimetic of a pre-fusion SNAREpin lacking the portion of the v-SNARE which zippers last to trigger fusion. The “central helix” of complexin is anchored to one SNARE complex while its “accessory helix” extends away at ~45° and bridges to a second complex, occupying the vacant v-SNARE binding site to inhibit fusion. That the accessory helix competes with the v-SNARE for t-SNARE binding was expected, but surprisingly, the interaction occurs inter-molecularly. Thus complexin organizes the SNAREs into a zig-zag topology which, when interposed between the vesicle and plasma membranes, is incompatible with fusion.

Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids, such as triacylglycerol (TG), as reservoirs of metabolic energy and membrane precursors. The Arf1/COPI protein machinery, known for its role in vesicle trafficking, regulates LD morphology, targeting of specific proteins to LDs and lipolysis through unclear mechanisms. Recent evidence shows that Arf1/COPI can bud nano-LDs (∼60 nm diameter) from phospholipid-covered oil/water interfaces in vitro. We show that Arf1/COPI proteins localize to cellular LDs, are sufficient to bud nano-LDs from cellular LDs, and are required for targeting specific TG-synthesis enzymes to LD surfaces. Cells lacking Arf1/COPI function have increased amounts of phospholipids on LDs, resulting in decreased LD surface tension and impairment to form bridges to the ER. Our findings uncover a function for Arf1/COPI proteins at LDs and suggest a model in which Arf1/COPI machinery acts to control ER-LD connections for localization of key enzymes of TG storage and catabolism.DOI: http://dx.doi.org/10.7554/eLife.01607.001

Membrane fusion occurs when SNAREpins fold up between lipid bilayers. How much energy is generated during SNAREpin folding and how this energy is coupled to the fusion of apposing membranes is unknown. We have used a surface forces apparatus to determine the energetics and dynamics of SNAREpin formation and characterize the different intermediate structures sampled by cognate SNAREs in the course of their assembly. The interaction energy-versus-distance profiles of assembling SNAREpins reveal that SNARE motifs begin to interact when the membranes are 8 nm apart. Even after very close approach of the bilayers (approximately 2-4 nm), the SNAREpins remain partly unstructured in their membrane-proximal region. The energy stabilizing a single SNAREpin in this configuration (35 k(B)T) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of pure lipid bilayers (40-50 k(B)T).

Oil-in-water emulsions are currently being investigated to facilitate the transport of viscous heavy oils. The behavior of these emulsions is largely controlled by the interfaces between oil drops and water. The surface-active components of crude oil, such as asphaltenes and naphthenic acids, compete among themselves at these interfaces and also with possibly added synthetic surfactant emulsifier. Here, we present a study of dynamic interfacial tension of interfaces between water and a model oil (toluene) in which variable amounts of asphaltenes are solubilized. We show that pH has a strong influence on interfacial properties of asphaltenes at the oil/water interface. At high or low pH, asphaltenes functional groups become charged, enhancing its surface activity. The influence of lower-molecular-weight surface-active species, such as the natural naphthenic acids contained in maltenes (crude oil without asphaltenes), has been investigated, and an interaction between asphaltenes and maltenes that facilitates molecular arrangement at the interface was detected. Several micropipette experiments, in which micrometric drops have been manipulated, are also described and indicate that very little coalescence of water droplets is observed at high or low pH. The microscopic properties of the interface and the macroscopic behavior of the emulsion are determined to be correlated.

The diverse structure and regulated deformation of lipid bilayer membranes are among a cell's most fascinating features. Artificial membrane-bound vesicles, known as liposomes, are versatile tools for modeling biological membranes and delivering foreign objects to cells. To fully mimic the complexity of cell membrane and optimize the efficiency of delivery vesicles, controlling liposome shape (both statically and dynamically) is of utmost importance. Here we report the assembly, arrangement, and remodeling of liposomes with designer geometry: all of which are exquisitely controlled by a set of modular, reconfigurable DNA nanocages. Tubular and toroidal shapes, among others, are transcribed from DNA cages to liposomes with high fidelity, giving rise to membrane curvatures present in cells yet previously difficult to construct in vitro. Moreover, the conformational changes of DNA cages drive membrane fusion and bending with predictable outcomes, opening up opportunities for the systematic study of membrane mechanics.Cells have evolved sophisticated mechanisms to regulate membrane shape and dynamics 1 . In the past few decades, scientists and engineers developed methods to generate artificial vesicles, or liposomes, as both model systems to study cell biology and drug carriers to interfere with cell behaviour 2,3 . The geometry of a liposome defines its physical and chemical properties (fusogenicity, binding affinity to proteins, susceptibility to enzymatic modifications, etc.), and therefore needs to be carefully controlled for each specific study and application. While existing techniques are capable of generating size-defined spherical liposomes and certain aspherical vesicles by means of bottom-up lipid assembly and mechanically distorting membranes 4-10 , design and experiment restraints (for example, lipid * Correspondence to: chenxiang.lin@yale.edu. Author contributions Z.Z. initiated the project, designed and carried out most of the experiments, analyzed the data, and prepared most of the manuscript. Y.Y. performed cryo-EM study and prepared the manuscript. F.P. modeled energy input for membrane fusion and prepared the manuscript. M.L. performed tomography study, analyzed the data, and prepared the manuscript. C.L. initiated the project, designed and supervised the study, interpreted the data, and prepared the manuscript. All authors reviewed and approved the manuscript. Competing financial interestsAuthors declare no competing financial interests. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript composition 11-15 ) often limit a method's adaptability, precision, and programmability. In this work, we take a bioinspired approach (namely DNA nanotemplating) to design, build, and change liposome shapes in a programmable, deterministic manner. Unlike previous methods that rely on trial and error to tune vesicle shape, here we directly programed the geometry of a liposome into its DNA template.Rapid development of DNA-origami technique has led to the construction o...