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Subcellular localization of proteins acting on the endomembrane system is primarily regulated via membrane trafficking. To obtain and maintain the correct protein composition of the plasma membrane and membrane-bound organelles, the loading of selected cargos into transport vesicles is critically regulated at donor compartments by adaptor proteins binding to the donor membrane, the cargo molecules and the coat-protein complexes, including the clathrin coat. The ANTH/ENTH/VHS domain-containing protein superfamily generally comprises a structurally related ENTH, ANTH, or VHS domain in the N-terminal region and a variable C-terminal region, which is thought to act as an adaptor during transport vesicle formation. This protein family is involved in various plant processes, including pollen tube growth, abiotic stress response and development. In this review, we provide an overview of the recent findings on ANTH/ENTH/VHS domain-containing proteins in plants.

To disentangle the elusive lipid-protein interactions in T-cell activation, we investigate how externally imposed variations in mobility of key membrane proteins (T-cell receptor [TCR], kinase Lck, and phosphatase CD45) affect the local lipid order and protein colocalisation. Using spectral imaging with polarity-sensitive membrane probes in model membranes and live Jurkat T cells, we find that partial immobilisation of proteins (including TCR) by aggregation or ligand binding changes their preference towards a more ordered lipid environment, which can recruit Lck. Our data suggest that the cellular membrane is poised to modulate the frequency of protein encounters upon alterations of their mobility, for example in ligand binding, which offers new mechanistic insight into the involvement of lipid-mediated interactions in membrane-hosted signalling events.

Plasma membrane carries out multiple physiological functions that require its dynamic and tightly regulated organization into specialized domains of different size, stability, and lipid/protein composition. Sphingolipids are a group of lipids in which the plasma membrane is particularly enriched, thus being crucial for its structure and function. A specific type of sphingolipid-enriched plasma membrane domains, where ergosterol is depleted and lipids are tightly packed in a rigid gel phase, has recently been found in several fungal species, including yeasts and moulds. After presenting the main biophysical features of gel domains and the experimental method for their detection in the fungal plasma membrane, we review these sphingolipid-enriched gel domains and illustrate their importance to both unicellular and multicellular fungi. First, the biophysical properties of the fungal sphingolipid-enriched domains will be analysed taking into consideration the plasma membrane sphingolipidome. Next, their possible biological roles will be summarized, including their relations with plasma membrane compartments and involvement in stress responses. Moreover, since the plasma membrane is a target for several antifungal compounds, a biophysical connection between sphingolipid-enriched domains and antifungal action will be explored.

To address biophysical principles and lipid interactions that underlie the properties of membrane proteins, modifications that vary the neighbors of tryptophan residues in the highly dynamic transmembrane helix of GW4,20 ALP23 (acetyl-GGAW4 A(LA)6 LAW20 AGA-amide) were examined using deuterium NMR spectroscopy. It was found that L5,19 GW4,20 ALP23, a sequence isomer of the low to moderately dynamic GW5,19 ALP23, remains highly dynamic. By contrast, a removal of W4 to produce F4,5 GW20 ALP23 restores a low level of dynamic averaging, similar to that of the F4,5 GW19 ALP23 helix. Interestingly, a high level of dynamic averaging requires the presence of both tryptophan residues W4 and W20, on opposite faces of the helix, and does not depend on whether residue 5 is Leu or Ala. Aspects of helix unwinding and potential oligomerization are discussed with respect to helix dynamic averaging and the locations of particular residues at a phosphocholine membrane interface.

Synaptic vesicle proteins, including N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs), Synaptotagmin‐1 and Complexin, are responsible for controlling the synchronised fusion of synaptic vesicles with the presynaptic plasma membrane in response to elevated cytosolic calcium levels. A range of structures of SNAREs and their regulatory proteins have been elucidated, but the exact organisation of these proteins at synaptic junction membranes remains elusive. Here, we have used cryoelectron tomography to investigate the arrangement of synaptic proteins in an in vitro reconstituted fusion system. We found that the separation between vesicle and target membranes strongly correlates with the organisation of protein complexes at junctions. At larger membrane separations, protein complexes assume a ‘clustered’ distribution at the docking site, inducing a protrusion in the target membrane. As the membrane separation decreases, protein complexes become displaced radially outwards and assume a ‘ring‐like’ arrangement. Our findings indicate that docked vesicles can possess a wide range of protein complex numbers and be heterogeneous in their protein arrangements.

Dimerization of the small GTPase Arf is prerequisite for the scission of COPI-coated transport vesicles. Here, we quantify the monomer/dimer equilibrium of Arf within the membrane and show that after membrane scission, Arf dimers are restricted to donor membranes. By hydrogen exchange mass spectrometry, we define the interface of activated dimeric Arf within its switch II region. Single amino acid exchanges in this region reduce the propensity of Arf to dimerize. We suggest a mechanism of membrane scission by which the dimeric form of Arf is segregated to the donor membrane. Our data are consistent with the previously reported absence of dimerized Arf in COPI vesicles and could explain the presence of one single scar-like noncoated region in each COPI vesicle.

Cell surface translocation of the chaperone glucose-regulated protein 78 kDa (GRP78) is a key event that promotes cancer cell survival during endoplasmic reticulum (ER) stress. Here, we identify Gα-interacting vesicle-associated protein (GIV) - an enhancer of prosurvival signaling during ER stress - as a binding partner of GRP78. We show that GIV and GRP78 interact in an ER stress-dependent manner through their respective carboxyl terminal domains and that GIV aids in the localization of GRP78 to the plasma membrane. Kaplan-Meier analysis of disease-free survival in cancer patients shows poor prognosis for patients with high expression of both GIV and GRP78, further suggesting a vital role for these two proteins in enhancing cancer cell viability.

The fibronectin type II (FnII) domain, present in diverse vertebrate proteins, plays crucial roles in several fundamental biological processes. PDC-109, the major bovine seminal plasma protein, contains two FnII domains that bind to choline phospholipids on sperm plasma membrane and induce lipid efflux crucial for successful fertilization. PDC-109 also exhibits chaperone-like activity and protects other proteins against various types of stress. Here, we show that a core tryptophan residue is highly conserved across species in the FnII domains. Mutation of conserved tryptophan residues W47, W93, and W106 in the FnII domains of PDC-109 to alanine leads to drastic decrease or complete abolition of membrane-binding and chaperone-like activities. These observations suggest that conserved tryptophans are important for the function of FnII proteins.

Phosphatidic acid (PA) is the simplest cellular glycerophospholipid characterized by unique biophysical properties: a small headgroup; negative charge; and a phosphomonoester group. Upon interaction with lysine or arginine, PA charge increases from -1 to -2 and this change stabilizes protein-lipid interactions. The biochemical properties of PA also allow interactions with lipids in several subcellular compartments. Based on this feature, PA is involved in the regulation and amplification of many cellular signalling pathways and functions, as well as in membrane rearrangements. Thereby, PA can influence membrane fusion and fission through four main mechanisms: it is a substrate for enzymes producing lipids (lysophosphatidic acid and diacylglycerol) that are involved in fission or fusion; it contributes to membrane rearrangements by generating negative membrane curvature; it interacts with proteins required for membrane fusion and fission; and it activates enzymes whose products are involved in membrane rearrangements. Here, we discuss the biophysical properties of PA in the context of the above four roles of PA in membrane fusion and fission.

The conserved oligomeric Golgi (COG) complex, a multisubunit tethering complex of the CATCHR (complexes associated with tethering containing helical rods) family, controls membrane trafficking and ensures Golgi homeostasis by orchestrating retrograde vesicle targeting within the Golgi. In humans, COG defects lead to severe multisystemic diseases known as COG‐congenital disorders of glycosylation (COG‐CDG). The COG complex both physically and functionally interacts with all classes of molecules maintaining intra‐Golgi trafficking, namely SNAREs, SNARE‐interacting proteins, Rabs, coiled‐coil tethers, and vesicular coats. Here, we review our current knowledge of COG‐related trafficking and glycosylation defects in humans and model organisms, and analyze possible scenarios for the molecular mechanism of the COG orchestrated vesicle targeting.

The Golgi apparatus is an important site for the modification of most secreted and membrane proteins. Glycan processing is the major class of modification and is mediated by a large number of Golgi-resident glycosyltransferases and glycosidases. These Golgi enzymes are largely type II transmembrane domain (TMD) proteins consisting of a short N-terminal cytosolic tail, a relatively short TMD and a lumenal 'stem/stalk' region which acts as a spacer between the catalytic domain and the lipid bilayer. The cytosolic tail, TMD, and stem together make what is termed the CTS domain which is responsible for the specific localisation of these enzymes within sub-Golgi compartments via multiple mechanisms. In addition, the catalytic domains of some Golgi enzymes are secreted as a consequence of proteolytic cleavage within their TMDs or stem regions. Finally, there is evidence to suggest that when the retention of Golgi enzymes is perturbed they are targeted for lysosomal degradation.

PI3KC2α is a phosphoinositide 3-kinase with a recently reported function in platelets; PI3KC2α-deficient mouse platelets have altered membrane structure and impaired function. Yet, how these membrane changes cause platelet dysfunction remains unknown. Here, focused ion beam-scanning electron microscopy of PI3KC2α-deficient platelet ultrastructure reveals a specific effect on the internal membrane structure, while liquid chromatography-tandem mass spectrometry profiling of 294 lipid species shows unaltered lipid composition. Functionally, PI3KC2α-deficient platelets exhibit impaired thrombosis specifically under conditions involving membrane tethering. These studies indicate that the structural changes in PI3KC2α-deficient platelets are limited to the membrane, occur without major changes in lipid composition, and selectively impair cell function during thrombus formation. These findings illustrate a unique mechanism that may be targetable for anti-thrombotic benefit.

Neurotransmitter and hormone release involve calcium-triggered fusion of a cargo-loaded vesicle with the plasma membrane. The initial connection between the fusing membranes, called the fusion pore, can evolve in various ways, including rapid dilation to allow full cargo release, slow expansion, repeated opening-closing, and resealing. Pore dynamics determine the kinetics of cargo release and the mode of vesicle recycling, but how these processes are controlled are poorly understood. Previous reconstitutions could not monitor single pores, limiting mechanistic insight they could provide. Recently developed nanodisc-based fusion assays allow reconstitution and monitoring of single pores with unprecedented detail and hold great promise for future discoveries. They recapitulate various aspects of exocytotic fusion pores, but comparison is difficult because different approaches suggested very different exocytotic fusion pore properties, even for the same cell type. In this Review, I discuss how most of the data can be reconciled, by recognizing how different methods probe different aspects of the same fusion process. The resulting picture is that fusion pores have broadly distributed properties arising from stochastic processes which can be modulated by physical constraints imposed by proteins, lipids and membranes.

The mechanisms by which exosomes (nano-vesicular messengers of cells) are taken up by recipient cells are poorly understood. We hypothesized that histones associated with these nano-particles are the ligands which facilitate their interaction with cell surface syndecan-4 (SDC4) to mediate their uptake. We show that the incubation with fetuin-A (exosome-associated proteins) and histones mediates the uptake of exosomes that are normally not endocytosed. Similarly, hydroxyapatite nanoparticles incubated with fetuin-A and histones (FNH) are internalized by tumor cells, while nanoparticles incubated with fetuin-A alone (FN) are not. The uptake of exosomes and FNH, both of which move to the perinuclear region of the cell, is attenuated in SDC4-knockdown cells. Data show that FNH can compete with exosomes for uptake and that both use SDC4 as uptake receptors.

Many human-cultured cell lines survive glucose starvation, but the underlying mechanisms remain unclear. Here, we searched for proteins required for cellular adaptation to glucose-limited conditions and identified several endoplasmic reticulum chaperones in the glucose-regulated protein (GRP) family as proteins enriched in the cellular membrane. Surprisingly, these proteins, which are required for cell surface localization of GLUT1 under high-glucose conditions, become dispensable for targeting GLUT1 to the surface upon glucose starvation. In marked contrast, cell surface localization of single-pass transmembrane proteins, such as epidermal growth factor receptor and CD98, is not disturbed by GRP78 depletion regardless of the extracellular glucose level. These results indicate that the extracellular glucose level regulates dependence on the GRPs for cell surface localization of multipass transmembrane proteins.

In order to probe the roles of PIP2 in the interactions between MIM I-BAR and model membranes, we performed a series of 10 μs-scale coarse-grained molecular dynamics simulations. Our results indicate that PIP2 plays predominant roles in the membrane binding of MIM I-BAR in a concentration-dependent manner and via electrostatic interactions. Besides, we find that the occurrence of the membrane curvature may induce the re-distribution of lipids in the membrane and result in the local enrichment of PIP2 at negatively curved membrane areas. Combining these roles of PIP2 in the membrane binding of MIM I-BAR helps explain how MIM I-BAR senses negative curvature and, thus, contributes to maintaining membrane protrusions.

Here, we investigated the role of the cell membrane protein CD146 in galectin-3-mediated endothelial cell migration at the molecular level. Our results show that knocking down CD146 significantly attenuates galectin-3-mediated cell migration. Pull-down assays, gel filtration, and biolayer interferometry further demonstrate that galectin-3 binds to the CD146 ectodomain (eFL) with a KD of ~1.1 μm. To identify the galectin-3-binding site, we used mass spectrometry to show that CD146 eFL has four N-glycosites, with PNGase F treatment indicating that N-glycans define the binding epitope. Galectin-3 likely interacts with Domain 5 on CD146 eFL, because it contains poly-N-acetyllactosamine sites, and deletion of this domain significantly reduces binding. Overall, our findings provide a better understanding of how galectin-3 interacts with cell membrane receptors to mediate endothelial cell migration.

Talins are cytoplasmic adapter proteins essential for integrin-mediated cell adhesion to the \ud extracellular matrix. Talins control the activation state of integrins, link integrins to cytoskeletal \ud actin, recruit numerous signalling molecules that mediate integrin signalling, and coordinate \ud recruitment of microtubules to adhesion sites via interaction with KANK (kidney ankyrin repeat-\ud containing) proteins. Vertebrates have two talin genes, TLN1 and TLN2. Although talin1 and \ud talin2 share 76% protein sequence identity (88% similarity), they are not functionally redundant, \ud and the differences between the two isoforms are not fully understood. In this Review, we focus \ud on the similarities and differences between the two talins in terms of structure, biochemistry \ud and function, which hint at subtle differences in fine-tuning adhesion signalling.

Omega-3 (ω-3) fatty acids (FAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are known to have important roles in human health and disease. Besides being utilized as fuel, ω-3 FAs have specific functions based on their structural characteristics. These functions include serving as ligands for several receptors, precursors of lipid mediators, and components of membrane glycerophospholipids (GPLs). Since ω-3 FAs (especially DHA) are highly flexible, the levels of DHA in GPLs may affect membrane biophysical properties such as fluidity, flexibility, and thickness. Here, we summarize some of the cellular mechanisms for incorporating DHA into membrane GPLs and propose biological effects and functions of DHA-containing membranes of several cell and tissue types. This article is protected by copyright. All rights reserved.

Ion channels facilitate diffusion of ions across cell membranes for such diverse purposes as neuronal signaling, muscular contraction, and fluid homeostasis. Solute transporters often utilize ionic gradients to move aqueous solutes up their concentration gradient, also fulfilling a wide variety of tasks. Recently, an increasing number of ion channel-transporter ('chansporter') complexes have been discovered. Chansporter complex formation may overcome what could otherwise be considerable spatial barriers to rapid signal integration and feedback between channels and transporters, the ions and other substrates they transport, and environmental factors to which they must respond. Here, current knowledge in this field is summarized, covering both heterologous expression structure/function findings and potential mechanisms by which chansporter complexes fulfill contrasting roles in cell signaling in vivo.

Plasmalogens are a unique class of phospholipids that are present in many organisms. Their presence in cell membranes has intrigued researchers for decades due to their unique molecular structure, namely the vinyl-ether bond at the sn-1 position, and their association with brain related disorders. Apparently, based on their amount in the cell membranes, their function is to provide exclusive structural and dynamical properties to these complex molecular assemblies. Yet, many of their physiological roles manifested through their biophysical properties have been challenging to identify. In this review, the biophysical properties of plasmalogens are discussed and compared to other lipid species. The role of plasmalogens is examined in the context of cell membrane function, and some future directions are given.

Plasmalogens, mostly ethanolamine-containing alkenyl ether phospholipids, are a major subclass of glycerophospholipids. Plasmalogen synthesis is initiated in peroxisomes and completed in the endoplasmic reticulum. The absence of plasmalogens in several organs of peroxisome biogenesis-defective patients suggests that the de novo synthesis of plasmalogens plays a pivotal role in its homeostasis in tissues. Plasmalogen synthesis is regulated by modulating the stability of fatty acyl-CoA reductase 1 on peroxisomal membranes, a rate-limiting enzyme in plasmalogen synthesis, by sensing plasmalogens in the inner leaflet of plasma membranes. Dysregulation of plasmalogen homeostasis impairs cholesterol biosynthesis by altering the stability of squalene monooxygenase, a key enzyme in cholesterol biosynthesis, implying physiological consequences of plasmalogen homeostasis with respect to cholesterol metabolism in cells, as well as in organs such as the liver.

[EN] We have identified in yeast a connection between two master regulators of cell growth: a biochemical connection involving the TORC1 protein kinase (which activates protein synthesis, nutrient uptake, and anabolism) and a biophysical connection involving the plasma membrane proton-pumping H+ -ATPase Pma1 (which drives nutrient and K+ uptake and regulates pH homeostasis). Raising the temperature to nonpermissive values in a TOR thermosensitive mutant decreases Pma1 activity. Rapamycin, a TORC1 inhibitor, inhibits Pma1 dependent on its receptor Fpr1 and on the protein phosphatase Sit4, a TORC1 effector. Mutation of either Sit4 or Tco89, a nonessential subunit of TORC1, decreases proton efflux, K+ uptake, intracellular pH, cell growth, and tolerance to weak organic acids. Tco89 does not affect Pma1 activity but activates K+ transport., We thank Prof. Michael N. Hall (Basel) for strains SH100 and SH221, and Prof. Joaquin Arino (Barcelona) for strain BY4741 and its null mutant derivatives fpr1 Delta 0, sit4 Delta 0, and tco89 Delta 0. We also thank Prof. Francisco Portillo (Madrid) for the antibody against the double phosphorylation of Ser911 Thr912 in Pma1. This work was funded by grant PROME-TEOII/2014/041 from Generalitat Valenciana (Valencia, Spain).

The role of myristoylation in the localization and catalytic activity of Src at focal adhesions was investigated by live-cell imaging and site-directed mutagenesis. Although the majority of activated Src molecules are localized at focal adhesions, it is unclear how activated Src molecules are recruited to focal adhesions. Because Src is activated at the cell membrane, translocation of Src to cell membranes is considered to be essential for its recruitment to focal adhesions. Membrane-targeting-deficient Src mutant SrcG2A localizes at focal adhesions, indicating direct recruitment of Src from cytosol to focal adhesions. Furthermore, directly recruited Src molecules are shown to enhance paxillin dynamics at focal adhesions. These results reveal that the regulation of Src activation and translocation is more complex than previously suggested.

Previous works proposed that aquaporins behave as mechanosensitive channels. However, principal issues about mechanosensitivity of aquaporins are not known. In this work, we characterized the mechanosensitive properties of the water channels BvTIP1;2 (TIP1) and BvPIP2;1 (PIP2) from red beet (Beta vulgaris). We simultaneously measured the mechanical behavior and the water transport rates during the osmotic response of emptied-out oocytes expressing TIP1 or PIP2. Our results indicate that TIP1 is a mechanosensitive aquaporin, whereas PIP2 is not. We found that a single exponential function between the osmotic permeability coefficient and the volumetric elastic modulus governs the mechanosensitivity of TIP1. Finally, homology modeling analysis indicates that putative residues involved in mechanosensitivity show different quantity and distribution in TIP1 and PIP2.

Many strains of Helicobacter pylori encode a protein secreting type IV secretion system called the Cag-T4SS. Here, we characterize one of the Cag-T4SS specific components, CagU (HP0531). We report that CagU is a bacterial inner membrane-associated protein, partially processed at the C-terminus, and that it interacts with the VirB6 and VirB8 homologues CagW and CagV, respectively. The level of expression of CagU is partially affected in the absence of cagX, cagW and cagV. Deletion of cagU aborts surface localization of CagA and affects the expression levels of CagI and CagH, which are involved in the Cag-T4SS pilus formation. Complementation of the cagU null mutation by wild-type cagU restores all these functions, suggesting its importance for Cag-T4SS function. This article is protected by copyright. All rights reserved.

A screen to identify lysosomal-expressed ion channels led to the discovery of the human Sidt2 protein. Sidt2 is expressed within lysosomal organelles but as a result of heterologous overexpression the protein is also detectable within the plasma membrane of human embryonic kidney cells. The overexpressed protein leads to cell depolarization upon sodium addition. Accordingly in whole-cell patch clamp experiments a spontaneous noninactivating monovalent cation current can be detected in Sidt2-overexpressing cells. Strong overexpression of Sidt2 in HEK293 cells is attended by a significant reduction/loss of detectable lysosomes, indicating that the overexpressed protein leads to lysosomal dysfunction, a hallmark of Alzheimer's disease. Sidt2 is located on chromosome 11q23, a locus repeatedly found by chromosomal mapping of Alzheimer's disease-related genes.

Non-erythroid spectrin or fodrin is present as part of the γ-tubulin ring complex (γ-TuRC) in brain tissue and brain derived cells. Here, we show that fodrin, which is otherwise known for providing structural support to the cell membrane, interacts directly with γ-tubulin within the γ-TuRC through a GRIP2-like motif. Turbidometric analysis of microtubule polymerization with nucleation-potent γ-TuRC isolated from HEK-293 cells that lack fodrin and the γ-TuRC from goat brain that contains fodrin shows inefficiency of the latter to promote nucleation. The involvement of fodrin was confirmed by the reduction in the microtubule polymerization efficiency of HEK-293 derived γ-TuRCs upon addition of purified brain fodrin. Thus, the interaction of fodrin with gamma-tubulin is responsible for its inhibitory effect on γ-tubulin mediated microtubule nucleation.

Connivance of cellular factors during virus-host cell membrane fusion is poorly understood. We have recently shown that cellular villin plays an important role during membrane fusion of reconstituted Sendai virosomes with hepatocytes. Here, we employed villin-null Chinese Hamster Ovary (CHO) cells, where villin expression led to an increased fusion with virosomes, which was further enhanced due to tyrosine phosphorylation in the presence of c-src. However, the villin RRI mutant, lacking actin-severing function, failed to augment membrane fusion. Furthermore, quantitative mass spectrometry and detailed analysis revealed Tyr499 to be the key phosphorylation site of villin responsible for the enhancement of virosome-CHO cell fusion. Overall, our results demonstrate a critical role for villin and its cell-type dependent phosphorylation in regulating membrane fusion.

In yeasts, the plasma membrane forms the barrier that protects the cell from the outside world, but also gathers and keeps valuable compounds inside. Although it is often suggested that hydrophobic molecules surpass this checkpoint by simple diffusion, it now becomes evident that protein-facilitated transport mechanisms allow for selective import and export of triglycerides, fatty acids, alkanes, and sterols in yeasts. During biomass production, hydrophobic carbon sources enter and exit the cell efficiently in a strictly regulated manner that helps avoid toxicity. Furthermore, various molecules, such as yeast pheromones, secondary metabolites and xenobiotics, are exported to ensure cell-cell communication, or increase chances of survival. This review summarizes the current knowledge on how hydrophobic compounds interact with protein-facilitated transport systems on the plasma membrane and how selective import and export across the yeast plasma membrane is achieved. Both the model organism Saccharomyces cerevisiae, as well as unconventional yeasts are discussed.

Escherichia coli HtpX is an M48 family zinc metalloproteinase located in the cytoplasmic membrane. Previous studies suggested that it is involved in the quality control of membrane proteins. However, its in vivo proteolytic function has not been characterized in detail, mainly because the physiological substrates have not been identified and no model substrate that allows sensitive detection of the protease activity is available. We constructed a new model substrate of HtpX and established an in vivo semiquantitative and convenient protease activity assay system for HtpX. This system enables detection of differential protease activities of HtpX mutants carrying mutations in conserved regions. This system would also be useful for investigating the functions of HtpX and its homologs in other bacteria.

Our study aims to investigate the physiological role of organophosphate hydrolase (OPH), hitherto known for its involvement in the degradation of organophosphate insecticides and nerve agents in Sphingobium fuliginis. We find that OPH exists as part of the TonB-dependent Transport system that is involved in nutrient transport across the bacterial outer membrane. OPH interacts physically with the Ton complex components ExbD and TonB. The surface-exposed arginine residues (R91 and R96) of OPH facilitate its interaction with ExbD. OPH is targeted to the inner membrane of Escherichia coli only when it is co-expressed with either ExbD or the ExbB/ExbD complex. In the absence of ExbD, OPH remains in the cytoplasm. Our findings suggest a role for OPH in outer membrane transport.

Live cell imaging can provide important information on cellular dynamics; however, the full utilisation of this technology has been hampered by the limitations of imaging reagents. Metal-based complexes have the potential to overcome many of the issues common to many current imaging agents. The rhenium (I)-based complex fac-[Re(CO)(3)(1,10-phenanthroline)(4-pyridyltetrazolate)], herein referred to as ReZolve-ER, shows promise as a live cell imaging agent with rapid cell uptake, low cytotoxicity, resistance to photobleaching and compatibility with multicolour imaging. ReZolve-ER localised to the nuclear membrane/endoplasmic reticulum (ER) and allowed the detection of exocytotic events at the plasma membrane. Thus, we present a new imaging agent for monitoring live cell events in real time, which is ideal for imaging either short- or long-time courses. Refereed/Peer-reviewed

International audience; LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a high-affinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/Forster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves.

Odor transduction in the cilia of olfactory sensory neurons involves several ATP-requiring enzymes. ATP is generated by glycolysis in the ciliary lumen, using glucose incorporated from surrounding mucus, and by oxidative phosphorylation in the dendrite. During prolonged stimulation, the cilia maintain ATP levels along their length, by unknown means. We used immunochemistry, RT-PCR, and immunoblotting to explore possible underlying mechanisms. We found the ATP-shuttles, adenylate and creatine kinases, capable of equilibrating ATP. We also investigated how glucose delivered by blood vessels in the olfactory mucosa reaches the mucus. We detected, in sustentacular and Bowman's gland cells, the crucial enzyme in glucose secretion glucose-6-phosphatase, implicating both cell types as putative glucose pathways. We propose a model accounting for both processes.

Membrane fusion and fission are fundamental processes in living organisms. Membrane fusion occurs through the formation of a fusion pore, which is the structure that connects two lipid membranes during their fusion. Fusion pores can form spontaneously, but cells endow themselves with a set of proteins that make the process of fusion faster and regulatable. The fusion pore starts with a narrow diameter and dilates relatively slowly; it may fluctuate in size or can even close completely, producing a transient vesicle fusion (kiss-and-run), or can finally expand abruptly to release all vesicle contents. A set of proteins control the formation, dilation, and eventual closure of the fusion pore and, therefore, the velocity at which the contents of secretory vesicles are released to the extracellular medium. Thus, the regulation of fusion pore expansion or closure is key to regulate the release of neurotransmitters and hormones. Here, we review the phases of the fusion pore and discuss the implications in the modes of exocytosis.

Neurotransmitter release occurs in the form of quantal events by fusion of secretory vesicles with the plasma membrane, and begins with the formation of a fusion pore that has a conductance similar to that of a large ion channel or gap junction. In this review, we propose mechanisms of fusion pore formation and discuss their implications for fusion pore structure and function. Accumulating evidence indicates a direct role of soluble N-ethylmaleimide-sensitive-factor attachment receptor proteins in the opening of fusion pores. Fusion pores are likely neither protein channels nor purely lipid, but are of proteolipidic composition. Future perspectives to gain better insight into the molecular structure of fusion pores are discussed.

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor (GPCR) activated by endogenous proteases, in particular, trypsin. Although regulators of G protein signaling (RGS) are known to inhibit GPCR/Gα-mediated signaling, their specific effects on PAR2 are poorly understood at present. Here, we use a bioluminescence resonance energy transfer technique to investigate whether RGS16 and RGS18 bind PAR2 in live cells to regulate PAR2/Gαi/o -mediated signaling. Notably, we find that RGS16 binds to PAR2 in the presence of Gαi while RGS18 does not interact with PAR2, regardless of the presence of Gα. Both RGS16 and RGS18 inhibit PAR2/Gαi/o -mediated signaling. To our knowledge, the current study is the first to highlight the effects of RGS proteins on PAR2-mediated signaling.

PB1F2 is a proapoptotic protein encoded by an alternative reading frame in the influenza A virus. Its accumulation accelerates mitochondrial fragmentation by decreasing the mitochondrial membrane potential following translocation into the mitochondrial inner membrane space, but the mechanistic underpinnings remain unclear. Herein, the PB1F2 from HK97 was expressed and purified in soluble form. The interaction between PB1F2 and the mitochondrial membrane were investigated using three membrane mimics, liposomes, bicelles, and nanodiscs. We show that the interactions between PB1F2 and membrane mimics depend on lipid type and are time- and dose-dependent. The primary membrane target of PB1F2 is phosphatidylcholine, the lipid that forms the major component of mitochondrial inner membranes. PB1F2 disrupts the integrity of lipid membranes by forming micelle-like PB1F2-lipid assemblies.

Synaptotagmin-1 (Syt1) is the primary calcium sensor (Ca