The GTPase dynamin assembles at the necks of budded vesicles in vivo and functions in membrane fission. We have developed fluid supported bilayers with excess membrane reservoir, (SUPER) templates, to assay vesicle formation and membrane fission. Consistent with previous studies, in the absence of GTP, dynamin assembles in spirals forming long membrane tubules. GTP addition triggers disassembly, but not membrane fission arguing against models that fission is mediated by concerted and global GTP-driven conformational changes. In contrast, under physiological conditions in the constant presence of GTP, dynamin mediates membrane fission. Under these conditions, fluorescently-labeled dynamin cooperatively organizes into self-limited assemblies that continuously cycle at the membrane and drive vesicle release. When visualized at the necks of emergent vesicles, self-limited dynamin assemblies display intensity fluctuations and persist for variable time periods before fission. Thus, self-limited assemblies of dynamin generated in the constant presence of GTP catalyze membrane fission.

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Biological membrane fission is conducted by protein-driven stress. To create such membrane stress the GTPase dynamin-1, protein orchestrating membrane fission in endocytosis, assembles into helical scaffolds that constrict the necks of endocytic vesicles. We found that under constant GTP turnover two-rung dynamin scaffold is sufficient to produce fission of lipid nanotubes. Analyzing membrane fission by short dynamin scaffolds, we reveal a catalytic cycle which translates constriction stresses into fission. Upon constriction, coordinated membrane wedging by the scaffold facilitates reversible merger of the inner leaflet of the nanotube, the hemifission. Modeling of this reversible step identifies a low-energy path based on geometric coupling of the scaffold and the membrane. The final translation of the metastable hemifission into complete fission is stochastically linked to disassembly of the scaffold. This catalytic conversion of localized stresses into membrane remodeling suggests a novel paradigm for fission and fusion of cellular membranes.

The serotonin(1A) (5-HT(1A)) receptor is an important member of the superfamily of seven-transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding activity and G-protein coupling of the bovine hippocampal 5-HT(1A) receptor by depleting cholesterol from native membranes using methyl-beta-cyclodextrin (MbetaCD). Removal of cholesterol from bovine hippocampal membranes using varying concentrations of MbetaCD results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT to 5-HT(1A) receptors. This is accompanied by alterations in binding affinity and sites obtained from analysis of binding data. Importantly, cholesterol depletion affected G-protein-coupling of the receptor as monitored by the GTP-gamma-S assay. The concomitant changes in membrane order were reported by changes in fluorescence polarization of membrane probes such as DPH and TMA-DPH, which are incorporated at different locations (depths) in the membrane. Replenishment of membranes with cholesterol led to recovery of ligand binding activity as well as membrane order to a considerable extent. Our results provide evidence, for the first time, that cholesterol is necessary for ligand binding and G-protein coupling of this important neurotransmitter receptor. These results could have significant implications in understanding the influence of the membrane lipid environment on the activity and signal transduction of other G-protein-coupled transmembrane receptors.

The importance of curvature as a structural feature of biological membranes has been recognized for many years and has fascinated scientists from a wide range of different backgrounds. On the one hand, changes in membrane morphology are involved in a plethora of phenomena involving the plasma membrane of eukaryotic cells, including endo- and exocytosis, phagocytosis and filopodia formation. On the other hand, a multitude of intracellular processes at the level of organelles rely on generation, modulation, and maintenance of membrane curvature to maintain the organelle shape and functionality. The contribution of biophysicists and biologists is essential for shedding light on the mechanistic understanding and quantification of these processes. Given the vast complexity of phenomena and mechanisms involved in the coupling between membrane shape and function, it is not always clear in what direction to advance to eventually arrive at an exhaustive understanding of this important research area. The 2018 Biomembrane Curvature and Remodeling Roadmap of Journal of Physics D: Applied Physics addresses this need for clarity and is intended to provide guidance both for students who have just entered the field as well as established scientists who would like to improve their orientation within this fascinating area.

1. Serotonin is an intrinsically fluorescent biogenic amine that acts as a neurotransmitter and is found in a wide variety of sites in the central and peripheral nervous system. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions. 2. Serotonin exerts its diverse actions by binding to distinct cell surface receptors which have been classified into many groups. The serotonin1A (5-HT1A) receptor is the most extensively studied of the serotonin receptors and belongs to the large family of seven transmembrane domain G-protein coupled receptors. 3. The tissue and sub-cellular distribution, structural characteristics, signaling of the serotonin1A receptor and its interaction with G-proteins are discussed. 4. The pharmacology of serotonin1A receptors is reviewed in terms of binding of agonists and antagonists and sensitivity of their binding to guanine nucleotides. 5. Membrane biology of 5-HT1A receptors is presented using the bovine hippocampal serotonin1A receptor as a model system. The ligand binding activity and G-protein coupling of the receptor is modulated by membrane cholesterol thereby indicating the requirement of cholesterol in maintaining the receptor organization and function. This, along with the reported detergent resistance characteristics of the receptor, raises important questions on the role of membrane lipids and domains in the function of this receptor.

In this study, we examined the importance of membrane ergosterol and sphingolipids in the drug susceptibilities of Candida albicans. We used three independent methods to test the drug susceptibilities of erg mutant cells, which were defective in ergosterol biosynthesis. While spot and filter disk assays revealed that erg2 and erg16 mutant cells of C. albicans became hypersensitive to almost all of the drugs tested (i.e., 4-nitroquinoline oxide, terbinafine, o-phenanthroline, itraconazole, and ketoconazole), determination of the MIC at which 80% of the cells were inhibited revealed more than fourfold increase in susceptibility to ketoconazole and terbinafine. Treatment of wild-type C. albicans cells with fumonisin B1 resulted in 45% inhibition of sphingolipid biosynthesis and caused cells to become hypersensitive to the above drugs. Although erg mutants displayed enhanced membrane fluidity and passive diffusion, these changes alone were not sufficient to elicit the observed hypersusceptibility phenotype of erg mutants. For example, the induction in vitro of a 12% change in the membrane fluidity of C. albicans cells by a membrane fluidizer, benzyl alcohol, did not affect the drug susceptibilities of Candida cells. Additionally, the surface localization of green fluorescent protein-tagged Cdr1p, a major drug efflux pump protein of C. albicans, revealed that any disruption in ergosterol and sphingolipid interactions also interfered with its proper surface localization and functioning. A 50% reduction in the efflux of the Cdr1p substrate, rhodamine 6G, in erg mutant cells or in cells with a reduced sphingolipid content suggested a strong correlation between these membrane lipid components and this major efflux pump protein. Taken together, the results of our study demonstrate for the first time that there is an interaction between membrane ergosterol and sphingolipids, that a reduction in the content of either of these two components results in a disruption of this interaction, and that this disruption has deleterious effects on the drug susceptibilities of C. albicans cells.

Dynamin-related protein 1 (Drp1) is essential for mitochondrial and peroxisomal fission. Recent studies propose that Drp1 does not sever but rather constricts mitochondrial membranes allowing dynamin 2 (Dnm2) to execute final scission. Here, we report that unlike Drp1, Dnm2 is dispensable for peroxisomal and mitochondrial fission, as these events occurred in Dnm2 knockout cells. Fission events were also observed in mouse embryonic fibroblasts lacking Dnm1, 2 and 3. Using reconstitution experiments on preformed membrane tubes, we show that Drp1 alone both constricts and severs membrane tubes. Scission required the membrane binding, self-assembling and GTPase activities of Drp1 and occurred on tubes up to 250 nm in radius. In contrast, Dnm2 exhibited severely restricted fission capacity with occasional severing of tubes below 50 nm in radius. We conclude that Drp1 has both membrane constricting and severing abilities and is the dominant dynamin performing mitochondrial and peroxisomal fission.