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Neurotoxic amyloid β-peptides are thought to be a causative agent of Alzheimer’s disease in humans. The production of amyloid β-peptides from amyloid precursor protein (APP) could be diminished by enhancing α-processing; however, the physical interactions between APP and α-secretases are not well understood. In this study, we employed super-resolution light microscopy to examine in cell-free plasma membranes the abundance and association of APP and α-secretases ADAM10 (a disintegrin and metalloproteinase) and ADAM17. We found that both secretase molecules localize similarly closely to APP (within ≤50 nm). However, when cross-linking APP with antibodies directed against the GFP tag of APP, in confocal microscopy, we observed that only ADAM10 coaggregated with APP. Furthermore, we mapped the involved protein domain by using APP variants with an exchanged transmembrane segment or lacking cytoplasmic/extracellular domains. We identified that the transmembrane domain of APP is required for association with α-secretases and, as analyzed by Western blot, for α-processing. We propose that the transmembrane domain of APP interacts either directly or indirectly with ADAM10, but not with ADAM17, explaining the dominant role of ADAM10 in α-processing of APP. Further understanding of this interaction may facilitate the development of a therapeutic strategy based on promoting APP cleavage by α-secretases. [ABSTRACT FROM AUTHOR]

Tetraspanins are a family of small proteins with four transmembrane segments (TMSs) playing multiple roles in human physiology. Nevertheless, we know little about the factors determining their structure. In the study at hand, we focus on the small intracellular loop (SIL) between TMS2 and TMS3. There we have identified a conserved five amino acid core region with three charged residues forming an M-shaped backbone, which we call M-motif. The M´s plane runs parallel to the membrane surface and the central amino acid constitutes the inter-helix turning point. At the second position of the M-motif, in tetraspanin crystal structures we identified a glutamate oriented towards a lysine in the juxtamembrane region of TMS1. Using Tspan17 as example, we find that by mutating either the glutamate or juxtamembrane-lysine, but not upon glutamate/lysine swapping, expression level, maturation and ER-exit are reduced. We conclude that the SIL is more than a short linking segment but propose it is involved in shaping the tertiary structure of tetraspanins. [ABSTRACT FROM AUTHOR]

Fluorescence microscopy is powerful for analyzing the composition and dynamics of cellular elements, but studying precise molecule patterns is precluded due to diffraction limited resolution. This barrier has been lifted now through several superresolution microscopy techniques. They revealed that proteins assemble in defined groups (clusters). A new challenge thus appears for the biologist: to find out whether clusters are molecular machines, stabilizers of defined protein conformations, or simply protein reservoirs. [ABSTRACT FROM AUTHOR]

Abstract: For membrane fusion to occur, opposed lipid bilayers initially establish a fusion pore, often followed by complete mixing of the fusing membranes. Contemporary views suggest that during fusion lipid bilayers are continuous passive platforms that are disrupted and remodeled by catalytic proteins. Some models propose that even the architecture and composition of the fusion pore might be dominated by proteins rather than lipids. Hence, lipids have no regulatory contribution to this process; they simply adapt their shape passively for filling space between otherwise autonomous protein machineries. However, an increasing number of experimental findings indicate that membrane fusion critically depends on a variety of lipids and lipid derivatives. Therefore, a purely proteocentric view describes fusion mechanisms insufficiently. Instead, lipids have functions probably at different levels, as (i) a general influence on the propensity of lipid bilayers to fuse, (ii) a role in recruiting exocytotic proteins to the plasma membrane, (iii) a role in organizing membrane domains for fusion and (iv) direct regulatory effects on fusion protein complexes. In this review we have made an attempt to bring together the large body of evidence supporting a major role for lipids in membrane fusion either directly or indirectly. [Copyright &y& Elsevier]

The original ‘lipid raft’ hypothesis proposed that lipid-platforms/rafts form in the exoplasmic plasmalemmal leaflet by tight clustering of sphingolipids and cholesterol. Their physical state, presumably similar to liquid-ordered phases in model membranes, would confer detergent resistance to rafts and enriched proteins therein. Based on this concept, detergent resistant membranes (DRMs) from solubilized cells were considered to reflect pre-existing ‘lipid rafts’ in live cells. To date, more than 200 proteins were found in DRMs including also members of the SNARE superfamily, which are small membrane proteins involved in intracellular fusion steps. Their raft association indicates that they are not uniformly distributed, and, indeed, microscopic studies revealed that SNAREs concentrate in submicrometre-sized, cholesterol-dependent clusters at which vesicles fuse. However, the idea that SNARE clusters are ‘lipid rafts’ was challenged, as they do not colocalize with raft markers, and SNAREs are excluded from liquid-ordered phases in model membranes. Independent from this disagreement, in recent years the solubilization criterion has been criticized for several reasons, calling for a more exact definition of rafts. At a recent consensus on a revised raft model, the term ‘lipid rafts’ was replaced by ‘membrane rafts’ that were defined as ‘small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes’. As a result, after dismissing the terms ‘detergent resistant’ and ‘liquid-ordered’, it now appears that SNARE clusters are bona fide‘membrane rafts’. [ABSTRACT FROM AUTHOR]

Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins mediate organelle fusion in the secretory pathway. Different fusion steps are catalyzed by specific sets of SNARE proteins. Here we have used the SNAREs mediating the fusion of early endosomes and exocytosis, respectively, to investigate how pairing specificity is achieved. Although both sets of SNAREs promiscuously assemble in vitro, there is no functional crosstalk. We now show that they not only colocalize to overlapping microdomains in the membrane of early endosomes of neuroendocrine cells, but also form cis-complexes promiscuously, with the proportion of the different complexes being primarily dependent on mass action. Addition of soluble SNARE molecules onto native membranes revealed preference for cognate SNAREs. Furthermore, we found that SNAREs are laterally segregated at endosome contact sites, with the exocytotic synaptobrevin being depleted. We conclude that specificity in endosome fusion is mediated by the following two synergistically operating mechanisms: (i) preference for the cognate SNARE in ‘trans’ interactions and (ii) lateral segregation of SNAREs, leading to relative enrichment of the cognate ones at the prospective fusion sites. [ABSTRACT FROM AUTHOR]

Tomosyn is a 130-kDa syntaxin-binding protein that contains a large N-terminal domain with WD40 repeats and a C-terminal domain homologous to R-SNAREs. Here we show that tomosyn forms genuine SNARE core complexes with the SNAREs syntaxin 1 and SNAP-25. In vitro studies with recombinant proteins revealed that complex formation proceeds from unstructured monomers to a stable four-helical bundle. The assembled complex displayed features typical for SNARE core complexes, including a profound hysteresis upon unfolding-refolding transitions. No stable complexes were formed between the SNARE motif of tomosyn and either syntaxin or SNAP-25 alone. Furthermore, both native tomosyn and its isolated C-terminal domain competed with synaptobrevin for binding to endogenous syntaxin and SNAP-25 on inside-out sheets of plasma membranes. Tomosyn-SNARE complexes were effectively disassembled by the ATPase N-ethylmaleimide-sensitive factor together with its cofactor α-SNAP. Moreover, the C-terminal domain of tomosyn was as effective as the cytoplasmic portion of synaptobrevin in inhibiting evoked exocytosis in a cell-free preparation derived from PC12 cells. Similarly, overexpression of tomosyn in PC12 cells resulted in a massive reduction of exocytosis, but the release parameters of individual exocytotic events remained unchanged. We conclude that tomosyn is a soluble SNARE that directly competes with synaptobrevin in the formation of SNARE complexes and thus may function in down-regulating exocytosis. [ABSTRACT FROM AUTHOR]

Membrane fusion, one of the most fundamental processes in life, occurs when two separate lipid membranes merge into a single continuous bilayer. Fusion reactions share common features, but are catalyzed by diverse proteins. These proteins mediate the initial recognition of the membranes that are destined for fusion and pull the membranes close together to destabilize the lipid/water interface and to initiate mixing of the lipids. A single fusion protein may do everything or assemblies of protein complexes may be required for intracellular fusion reactions to guarantee rigorous regulation in space and time. Cellular fusion machines are adapted to fit the needs of different reactions but operate by similar principles in order to achieve merging of the bilayers. [Copyright &y& Elsevier]

During exocytosis, secretory granules fuse with the plasma membrane and discharge their content into the extracellular space. The exocytosed membrane is then reinternalized in a coordinated fashion. A role of clathrin-coated vesicles in this process is well established, whereas the involvement of a direct retrieval mechanism (often called kiss and run) is still debated. Here we report that a significant population of docked secretory granules in the neuroendocrine cell line PC12 fuses with the plasma membrane, takes up fluid-phase markers, and is retrieved at the same position. Fusion allows for complete discharge of small molecules, whereas GFP-labeled neuropeptide Y (molecular mass ≈35 kDa) is only partially released. Retrieved granules were preferentially associated with dynamin. Furthermore, recapture is inhibited by guanosine 5'-[γ,-thio]triphosphate and peptides known to block dynamin function. We conclude that secretory granules can be recaptured immediately after formation of an exocytotic opening by an endocytic reaction that is spatially and temporally coupled to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent fusion, but is not a reversal of the fusion reaction. [ABSTRACT FROM AUTHOR]

Examines the activity of the endogeneous vesicle-bound soluble NSF attachment protein receptor (SNARE) based on the binding of labeled recombinant SNARE to inside-out carrier supported plasma membrane sheets of PC12 cells. Downregulation by control proteins; Cell culture and transfection; Quantitation of fluorescence signals.

During exocytosis, SNARE proteins of secretory vesicles interact with the corresponding SNARE proteins in the plasmalemma to initiate the fusion reaction. However, it is unknown whether SNAREs are uniformly distributed in the membrane or whether specialized fusion sites exist. Here we report that in the plasmatemma, syntaxins are concentrated in 200 nm large, cholesterol-dependent clusters at which secretory vesicles preferentially dock and fuse. The syntaxin clusters are distinct from cholesterol- dependent membrane rafts since they are Triton X-100-soluble and do not co-patch with raft markers. Synaptosomal-associated protein (SNAP)-25 is also clustered in spots, which partially overlap with syntaxin. Cholesterol depletion causes dispersion of these clusters, which is associated with a strong reduction in the rate of secretion, whereas the characteristics of individual exocytic events are unchanged. This suggests that high local concentrations of SNAREs are required for efficient fusion. [ABSTRACT FROM AUTHOR]

In the February 8th issue of Cell, Wang et al. report the surprising finding that vacuolar fusion occurs at the periphery of the contact area of the vacuoles and not by the expansion of a central fusion pore. During fusion, a disk of boundary membrane is excised and left behind within the fused vacuoles. [Copyright &y& Elsevier]

Biochemical approaches revealed that tetraspanins are multi-regulatory proteins forming a web, where they act in tetraspanin-enriched-microdomains (TEMs). A microscopic criterion differentiating between web and TEMs is lacking. Using super-resolution microcopy, we identify co-assemblies between the tetraspanins CD9 and CD81 and CD151 and CD81. CD9 assemblies contain as well the CD9/CD81-interaction partner EWI-2. Moreover, CD9 clusters are proximal to clusters of the CD81-interaction partner CD44 and CD81-/EWI-2-interacting ezrin–radixin–moesin proteins. Assemblies scatter unorganized across the cell membrane; yet, upon EWI-2 elevation, they agglomerate into densely packed arranged-crowds in a process independent from actin dynamics. In conclusion, microscopic clusters are equivalent to biochemical tetraspanin-assemblies, defining in their entirety the tetraspanin web. Cluster-agglomeration enriches tetraspanins, which makes agglomerations to a microscopic complement of TEMs. The microscopic classification of tetraspanin assemblies advances our understanding of this enigmatic protein family, whose members play roles in a plethora of cellular functions, diseases, and pathogen infections. [ABSTRACT FROM AUTHOR]

Members of the tetraspanin family have been identified as essential cellular membrane proteins in infectious diseases by nearly all types of pathogens. The present review highlights recently published data on the role of tetraspanin CD151, CD81, and CD63 and their interaction partners in host cell entry by human cytomegalo- and human papillomaviruses. Moreover, we discuss a model for tetraspanin assembly into trafficking platforms at the plasma membrane. These platforms might persist during intracellular viral trafficking. [ABSTRACT FROM AUTHOR]

Recently, biomaterials have been widely used in a variety of medical applications. We previously reported that a poly-l-lactic acid (PLLA) nanosheet shows anti-adhesive properties and constitutes a useful biomaterial for preventing unwanted wound adhesion in surgical operations. In this article, we examine whether the PLLA nanosheet can be specifically modified with biomacromolecules on one surface only. Such an approach would endow each side of the nanosheet with discrete functions, that is anti-adhesive and pro-healing properties. We fabricated two distinct PLLA nanosheets: (i) collagen cast on the surface of a PLLA nanosheet (Col-Cast-PLLA) and (ii) collagen spin-coated on the nanosheet (Col-Spin-PLLA). In the Col-Spin-PLLA nanosheet, the collagen layer had a thickness of 5–10 nm on the PLLA surface and displayed increased hydrophilicity compared to both PLLA and Col-Cast-PLLA nanosheets. In addition, atomic force microscopy showed disorganized collagen fibril formation on the PLLA layer when covered using the spin-coating method, while apparent bundle formations of collagen were formed in the Col-Cast-PLLA nanosheet. The Col-Spin-PLLA nanosheet provided a microenvironment for cells to adhere and spread. By contrast, the Col-Cast-PLLA nanosheet displayed reduced cell adhesion compared to the Col-Spin-PLLA nanosheet. Consistent with these findings, immunocytochemical analysis clearly showed fine networks of actin filaments in cells cultured on the Col-Spin-PLLA, but not the Col-Cast-PLLA nanosheet. Therefore, the Col-Spin-PLLA nanosheet was shown to be more suitable for acting as a scaffold. In conclusion, we have succeeded in developing a heterofunctional nanosheet comprising a collagen modified side, which has the ability to rapidly adhere cells, and an unmodified side, which acts as an adhesion barrier, by using a spin-coating technique. [ABSTRACT FROM AUTHOR]

Munc-18 interacts with the SNARE protein syntaxin and is supposed to influence transmitter release by controlling the formation of exocytosis-relevant SNARE complexes. Here, we used combined biochemical and physiological analyses to study the role of the Munc-18/syntaxin interaction in large dense core vesicle (LDCV) exocytosis of neuroendocrine PC12 cells. We compared two Munc-18 mutants carrying mutations in the syntaxin-binding region and show that Munc-18's membrane association depends on direct binding to syntaxin. The data suggest that perturbation of syntaxin binding inhibits neurotransmitter release upstream of the individual fusion event implying an essential role of the Munc-18/syntaxin complex leading to exocytosis. Furthermore, we show that a Munc-18 mutant lacking any syntaxin binding has a stimulatory effect on secretion, and provide evidence that the Munc-18/Mint1 interaction may constitute a second pathway for Munc-18 to regulate exocytosis. We propose that Munc-18 represents a dynamic link between syntaxin-related and Mint1-related mechanisms, both involved in the control of LDCV exocytosis in neuroendocrine cells. [ABSTRACT FROM AUTHOR]

During exocytosis, certain phospholipids may act as regulators of secretion. Here, we used several independent approaches to perturb the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] level in bovine chromaffin cells to investigate how changes of plasmalemmal PI(4,5)P2 affect secretion. Membrane levels of PI(4,5)P2 were estimated by analyzing images of lawns of plasma membranes labeled with fluorescent probes specific for PI(4,5)P2. The specific PI(4,5)P2 signal was enriched in submicrometer-sized clusters. In parallel patch-clamp experiments on intact cells, we measured the secretion of catecholamines. Overexpression of phosphatidylinositol-4-phosphate-5-kinase Iγ or infusion of PI(4,5)P2 through the patch pipette, increased the PI(4,5)P2 level in the plasma membrane and potentiated secretion. Expression of a membrane-targeted inositol 5-phosphatase domain of synaptojanin 1 eliminated PI(4,5)P2 from the membrane and abolished secretion. An inhibitor of phosphatidylinositol-3 kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyyran-4-one, led to a transient increase in the PI(4,5)P2 level that was associated with a potentiation of secretion. After prolonged incubation, the level of PI(4,5)P2 decreased and secretion was inhibited. Kinetic analysis showed that changes in PI(4,5)P2 levels led to correlated changes in the size of two releasable vesicle pools, whereas their fusion kinetics remained unaffected. We conclude that during both short- and long-term manipulations of PI(4,5)P2 level secretion scales with plasma membrane PI(4,5)P2 content and that PI(4,5)P2 has an early effect on secretion by regulating the number of vesicles ready for release. [ABSTRACT FROM AUTHOR]

SNARE proteins have been described as the effectors of fusion events in the secretory pathway more than two decades ago. The strong interactions between SNARE domains are clearly important in membrane fusion, but it is unclear whether they are involved in any other cellular processes. Here, we analyzed two classical SNARE proteins, syntaxin 1A and SNAP25. Although they are supposed to be engaged in tight complexes, we surprisingly find them largely segregated in the plasma membrane. Syntaxin 1A only occupies a small fraction of the plasma membrane area. Yet, we find it is able to redistribute the far more abundant SNAP25 on the mesoscale by gathering crowds of SNAP25 molecules onto syntaxin clusters in a SNARE-domain-dependent manner. Our data suggest that SNARE domain interactions are not only involved in driving membrane fusion on the nanoscale, but also play an important role in controlling the general organization of proteins on the mesoscale. Further, we propose these mechanisms preserve active syntaxin 1A-SNAP25 complexes at the plasma membrane. [ABSTRACT FROM AUTHOR]

Tetraspanins are master organizers of the cell membrane. Recent evidence suggests that tetraspanins themselves may become crowded by virus particles and that these crowds/aggregates co-internalize with the viral particles. Using microscopy, we studied human papillomavirus (HPV) type 16-dependent aggregates on the cell surface of tetraspanin overexpressing keratinocytes. We find that aggregates are (1) rich in at least two different tetraspanins, (2) three-dimensional architectures extending up to several micrometers into the cell, and (3) decorated intracellularly by filamentous actin. Moreover, in cells not overexpressing tetraspanins, we note that obscurin-like protein 1 (OBSL1), which is thought to be a cytoskeletal adaptor, associates with filamentous actin. We speculate that HPV contact with the cell membrane could trigger the formation of a large tetraspanin web. This web may couple the virus contact site to the intracellular endocytic actin machinery, possibly involving the cytoskeletal adaptor protein OBSL1. Functionally, such a tetraspanin web could serve as a virus entry platform, which is co-internalized with the virus particle. [ABSTRACT FROM AUTHOR]

During cell invasion, human papillomaviruses use large CD151 patches on the cell surface. Here, we studied whether these patches are defined architectures with features for virus binding and/or internalization. Super-resolution microscopy reveals that the patches are assemblies of closely associated nanoclusters of CD151, integrin α3 and integrin α6. Integrin α6 is required for virus attachment and integrin α3 for endocytosis. We propose that CD151 organizes viral entry platforms with different types of integrin clusters for different functionalities. Since numerous viruses use tetraspanin patches, we speculate that this building principle is a blueprint for cell-surface architectures utilized by viral particles. [ABSTRACT FROM AUTHOR]

Herpes simplex virus (HSV)-encoded glycoprotein B (gB) is the most abundant protein in the viral envelope and promotes fusion of the virus with the cellular membrane. In the present study, we found that gB impacts on the major histocompatibility complex (MHC)-II pathway of antigen presentation by fostering homotypic fusion of early endosomes and trapping MHC-II molecules in these altered endosomes. By using an overexpression approach, we demonstrated that transient expression of gB induces giant vesicles of early endosomal origin, which contained Rab5, early endosomal antigen 1 (EEA1), and large amounts of MHC-II molecules [human leukocyte antigen (HLA)-DR, and HLA-DM], but no CD63. In HSV-1-infected and stably transfected cell lines that expressed lower amounts of gB, giant endosomes were not observed, but strongly increased amounts of HLA-DR and HLA-DM were found in EEA1+ early endosomes. We used these giant vesicles as a model system and revealed that gB interacts with Rab5 and EEA1, and that gB-induced homotypic fusion of early endosomes to giant endosomes requires phosphatidylinositol 3-phosphate, the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptors, and the cytosolic gB sequence 889YTQVPN894. We conclude that gB expression alters trafficking of molecules of the HLA-II processing pathway, which leads to increased retention of MHC-II molecules in early endosomal compartments, thereby intercepting antigen presentation.--Niazy, N., Temme, S., Bocuk, D., Giesen, C., König, A., Temme, N., Ziegfeld, A., Gregers, T. F., Bakke, O., Lang, T., Eis-Hübinger, A. M., Koch, N. Misdirection of endosomal trafficking mediated by herpes simplex virus-encoded glycoprotein B. [ABSTRACT FROM AUTHOR]

The SNAREs SNAP25 and SNAP23 are proteins that are initially cytosolic after translation, but then become stably attached to the cell membrane through palmitoylation of cysteine residues. For palmitoylation to occur, membrane association is a prerequisite, but it is unclear which motif may increase the affinities of the proteins for the target membrane. In experiments with rat neuroendocrine cells, we find that a few basic amino acids in the cysteine-rich region of SNAP25 and SNAP23 are essential for plasma membrane targeting. Reconstitution of membrane-protein binding in a liposome assay shows that the mechanism involves protein electrostatics between basic amino acid residues and acidic lipids such as phosphoinositides that play a primary role in these interactions. Hence, we identify an electrostatic anchoring mechanism underlying initial plasma membrane contact by SNARE proteins, which subsequently become palmitoylated at the plasma membrane. [ABSTRACT FROM AUTHOR]

Non-vesicular lipid transport steps play a crucial role in lipid trafficking and potentially include spontaneous exchange. Since membrane contact facilitates this lipid transfer, it is most likely to occur at membrane contact sites (MCS). However, to date it is unknown whether closely attached biological membranes exchange lipids spontaneously. We have set up a system for studying the exchange of lipids at MCS formed between the endoplasmic reticulum (ER) and the plasma membrane. Contact sites were stably anchored and the lipids cholesterol and phosphatidylcholine (PC) were not capable of transferring spontaneously into the opposed bilayer. We conclude that physical contact between two associated biological membranes is not sufficient for transfer of the lipids PC and cholesterol. [ABSTRACT FROM AUTHOR]

Summary The molecular mechanisms regulating antigen translocation into the cytosol for cross-presentation are under controversial debate, mainly because direct data is lacking. Here, we have provided direct evidence that the activity of the endoplasmic reticulum (ER) translocon protein Sec61 is essential for endosome-to-cytosol translocation. We generated a Sec61-specific intrabody, a crucial tool that trapped Sec61 in the ER and prevented its recruitment into endosomes without influencing Sec61 activity and antigen presentation in the ER. Expression of this ER intrabody inhibited antigen translocation and cross-presentation, demonstrating that endosomal Sec61 indeed mediates antigen transport across endosomal membranes. Moreover, we showed that the recruitment of Sec61 toward endosomes, and hence antigen translocation and cross-presentation, is dependent on dendritic cell activation by Toll-like receptor (TLR) ligands. These data shed light on a long-lasting question regarding antigen cross-presentation and point out a role of the ER-associated degradation machinery in compartments distinct from the ER. [ABSTRACT FROM AUTHOR]

The initiation of adaptive immunity requires cell-to-cell contact between T cells and antigen-presenting cells. Together with immediate TCR signal transduction, the formation of an immune synapse (IS) is one of the earliest events detected during T cell activation. Here, we show that interaction of liver sinusoidal endothelial cells (LSEC) with naive CD8 T cells, which induces CD8 T cells without immediate effector function, is characterized by a multi-focal type IS. The co-inhibitory molecule B7H1, which is pivotal for the development of non-responsive LSEC-primed T cells, did not alter IS structure or TCRβ/CD11a cluster size or density, indicating that IS form does not determine the outcome of LSEC-mediated T cell activation. Instead, PD-1 signaling during CD8 T cell priming by LSEC repressed IL-2 production as well as sustained CD25 expression. When acting during the first 24 h of LSEC/CD8 T cell interaction, CD28 co-stimulation inhibited the induction of non-responsive LSEC-primed T cells. However, after more than 36 h of PD-1 signaling, CD28 co-stimulation failed to rescue effector function in LSEC-primed T cells. Together, these data show that during LSEC-mediated T cell priming, integration of co-inhibitory PD-1 signaling over time turns on a program for CD8 T cell development, that cannot be overturned by co-stimulatory signals. [ABSTRACT FROM AUTHOR]

Lipid transfer proteins (LTPs) are emerging as key players in lipid homeostasis by mediating non-vesicular transport steps between two membrane surfaces. Little is known about the driving force that governs the direction of transport in cells. Using the soluble LTP glycolipid transfer protein (GLTP), we examined GM1 (monosialotetrahexosyl-ganglioside) transfer to native membrane surfaces. With artificial GM1 donor liposomes, GLTP can be used to increase glycolipid levels over natural levels in either side of the membrane leaflet, i.e., external or cytosolic. In a system with native donor- and acceptor-membranes, we find that GLTP balances highly variable GM1 concentrations in a population of membranes from one cell type, and in addition, transfers lipids between membranes from different cell types. Glycolipid transport is highly efficient, independent of cofactors, solely driven by the chemical potential of GM1 and not discriminating between the extra- and intracellular membrane leaflet. We conclude that GLTP mediated non-vesicular lipid trafficking between native membranes is driven by simple thermodynamic principles and that for intracellular transport less than 1 µM GLTP would be required in the cytosol. Furthermore, the data demonstrates the suitability of GLTP as a tool for artificially increasing glycolipid levels in cellular membranes. [ABSTRACT FROM AUTHOR]

CD81 (TAPA-1) is a member of the widely expressed and evolutionary conserved tetraspanin family that forms complexes with a variety of other cell surface receptors and facilitates hepatitis C virus entry. Here, we show that CD81 is specifically required for the formation of lamellipodia in migrating dendritic cells (DCs). Mouse CD81-/- DCs, or murine and human CD81 RNA interference knockdown DCs lacked the ability to form actin protrusions, thereby impairing their motility dramatically. Moreover, we observed a selective loss of Rac1 activity in the absence of CD81, the latter of which is exclusively required for integrin-dependent migration on 2-dimensional substrates. Neither integrin affinity for substrate nor the size of basal integrin clusters was affected by CD81 deficiency in adherent DCs. However, the use of total internal reflection fluorescence microscopy revealed an accumulation of integrin clusters above the basal layer in CD81 knockdown cells. Furthermore, β1- or β2-integrins, actin, and Rac are strongly colocalized at the leading edge of DCs, but the very fronts of these cells protrude CD81-containing membranes that project outward from the actin-integrin area. Taken together, these data suggest a thus far unappreciated role for CD81 in the mobilization of preformed integrin clusters into the leading edge of migratory DCs on 2-dimensional surfaces. [ABSTRACT FROM AUTHOR]

Membrane proteins and membrane lipids are frequently organized in submicron-sized domains within cellular membranes. Factors thought to be responsible for domain formation include lipid-lipid interactions, lipid-protein interactions and protein-protein interactions. However, it is unclear whether the domain structure is regulated by other factors such as divalent cations. Here, we have examined in native plasma membranes and intact cells the role of the second messenger Ca2+ in membrane protein organization. We find that Ca2+ at low micromolar concentrations directly redistributes a structurally diverse array of membrane proteins via electrostatic effects. Redistribution results in a more clustered pattern, can be rapid and triggered by Ca2+ influx through voltage-gated calcium channels and is reversible. In summary, the data demonstrate that the second messenger Ca2+ strongly influences the organization of membrane proteins, thus adding a novel and unexpected factor that may control the domain structure of biological membranes. [ABSTRACT FROM AUTHOR]

We demonstrate three-dimensional (3D) super-resolution imaging of stochastically switched fluorophores distributed across whole cells. By evaluating the higher moments of the diffraction spot provided by a 4Pi detection scheme, single markers can be simultaneously localized with <10 nm precision in three dimensions in a layer of 650 nm thickness at an arbitrarily selected depth in the sample. By splitting the fluorescence light into orthogonal polarization states, our 4Pi setup also facilitates the 3D nanoscopy of multiple fluorophores. Offering a combination of multicolor recording, nanoscale resolution and extended axial depth, our method substantially advances the noninvasive 3D imaging of cells and of other transparent materials. [ABSTRACT FROM AUTHOR]

Summary: Signaling by ErbB receptors requires the activation of their cytoplasmic kinase domains, which is initiated by ligand binding to the receptor ectodomains. Cytoplasmic factors contributing to the activation are unknown. Here we identify members of the cytohesin protein family as such factors. Cytohesin inhibition decreased ErbB receptor autophosphorylation and signaling, whereas cytohesin overexpression stimulated receptor activation. Monitoring epidermal growth factor receptor (EGFR) conformation by anisotropy microscopy together with cell-free reconstitution of cytohesin-dependent receptor autophosphorylation indicate that cytohesins facilitate conformational rearrangements in the intracellular domains of dimerized receptors. Consistent with cytohesins playing a prominent role in ErbB receptor signaling, we found that cytohesin overexpression correlated with EGF signaling pathway activation in human lung adenocarcinomas. Chemical inhibition of cytohesins resulted in reduced proliferation of EGFR-dependent lung cancer cells in vitro and in vivo. Our results establish cytohesins as cytoplasmic conformational activators of ErbB receptors that are of pathophysiological relevance. [Copyright &y& Elsevier]

SNAREs are clustered membrane proteins essential for intracellular fusion steps. During fusion, three to four SNAREs with a Qa-, Qb-, Qc- and R-SNARE-motif form a complex. The core complex represents a QaQbQcR-SNARE-motif bundle, most certainly assembling in steps. However, to date it is unknown which intermediate SNARE complex observed in vitro also exists in vivo. Here we have applied comparative fluorescence recovery after photobleaching (FRAP)-studies as a novel approach for studying in intact cells a SNARE interaction involved in synaptic vesicle fusion [catalyzed by syntaxin 1A (Qa), SNAP25 (Qb/Qc) and synaptobrevin 2 (R)]. We find that the Qb-SNARE-motif of SNAP25 interacts reversibly with clustered syntaxin. The interaction requires most of the alpha helical Qb-SNARE-motif and depends on its position within the molecule. We conclude that a zippered QaQb-SNARE complex represents a short-lived SNARE intermediate in intact cells, most likely providing an initial molecular platform toward membrane fusion. [ABSTRACT FROM AUTHOR]

The soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein (SNAP) receptor (SNARE) protein syntaxin 1A forms nanosized clusters (membrane rafts) on the plasma membrane (PM) that are in equilibrium with freely diffusing syntaxin molecules. SNARE complex formation between syntaxin 1A and SNAP-25 (synaptosome-associated protein of 25 kDa) on the PM and synaptobrevin 2 on the vesicles (trans-SNAREs) is crucial for vesicle priming and fusion. This process might be impeded by the spontaneous accumulation of non-fusogenic cis-SNARE complexes formed when all three SNARE proteins reside on the PM. Weinvestigated the kinetics of cis-SNARE complex assembly and disassembly and both exhibited biphasic behavior. The experimental measurements were analyzed through integration of differential rate equations pertinent to the reaction mechanism and through the application of a heuristic search for time constants and concentrations using a genetic algorithm. Reconstruction of the measurements necessitated the partitioning of syntaxin into two phases that might represent the syntaxin clusters and free syntaxin outside the clusters. The analysis suggests that most of the syntaxin in the clusters is concentrated in a nonreactive form. Consequently, cis-SNARE complex assembly in the clusters is substantially slower than outside the rafts. Interestingly, the clusters also mediate efficient disassembly of cis-SNARE complexes possibly attributable to the high local concentration of complexes in the clusters area that allows efficient disassembly by the enzymatic reaction of NSF. [ABSTRACT FROM AUTHOR]

Myelin basic protein (MBP) is an essential structural component of CNS myelin. The electrostatic association of this positively charged protein with myelin-forming membranes is a crucial step in myelination, but the mechanism that regulates myelin membrane targeting is not known. Here, we demonstrate that phosphatidylinositol 4,5-bisphosphate (PIP2) is important for the stable association of MBP with cellular membranes. In oligodendrocytes, overexpression of synaptojanin 1-derived phosphoinositide 5-phosphatase, which selectively hydrolyzes membrane PIP2, causes the detachment of MBP from the plasma membrane. In addition, constitutively active Arf6/ Q67L induces the formation of PIP2-enriched endosomal vacuoles, leading to the redistribution of MBP to intracellular vesicles. Fluorescence resonance energy transfer imaging revealed an interaction of the PIP2 sensing probe PH-PLCδ1 with wild-type MBP, but not with a mutant MBP isoform that fails to associate with the plasma membrane. Moreover, increasing intracellular Ca2+, followed by phospholipase C-mediated PIP2 hydrolysis, as well as reduction of the membrane charge by ATP depletion, resulted in the dissociation of MBP from the glial plasma membrane. When the corpus callosum of mice was analyzed in acute brain slices by electron microscopy, the reduction of membrane surface charge led to the loss of myelin compaction and rapid vesiculation. Together, these results establish that PIP2 is an essential determinant for stable membrane binding of MBP and provide a novel link between glial phosphoinositol metabolism and MBP function in development and disease. [ABSTRACT FROM AUTHOR]

Abstract: Mild sonication of eukaryotic cells produces native plasma membrane sheets that retain their docked organelles, cytoskeleton structures and cytoplasmic complexes. While the delicate organization of membranous protein complexes remains undisturbed, their inner plasmalemmel leaflet can be rapidly exposed to bathing solutions, enabling specific biochemical manipulations. Here, we apply this system to track membrane-biochemistry kinetics. We monitor soluble NSF-attachment protein receptor (SNARE) complex assembly and disassembly on the plasma membrane at high time resolution. The results suggest two-phase kinetics for the assembly process and dependence of the disassembly kinetics on both N-ethyl maleimide-sensitive factor (NSF) and soluble NSF-attachment protein (-SNAP) concentrations. [Copyright &y& Elsevier]

Most plasmalemmal proteins organize in submicrometer-sized clusters whose architecture and dynamics are still enigmatic. With syntaxin 1 as an example, we applied a combination of far-field optical nanoscopy, biochemistry, fluorescence recovery after photobleaching (FRAP) analysis, and simulations to show that clustering can be explained by self-organization based on simple physical principles. On average, the syntaxin clusters exhibit a diameter of 50 to 60 nanometers and contain 75 densely crowded syntaxins that dynamically exchange with freely diffusing molecules. Self-association depends on weak homophilic protein-protein interactions. Simulations suggest that clustering immobilizes and conformationally constrains the molecules. Moreover, a balance between self-association and crowding- induced steric repulsions is sufficient to explain both the size and dynamics of syntaxin clusters and likely of many oligomerizing membrane proteins that form supramolecular structures. [ABSTRACT FROM AUTHOR]

Munc18–1, a protein essential for regulated exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like (SM) proteins. In vitro, Munc18–1 forms a tight complex with the SNARE syntaxin 1, in which syntaxin is stabilized in a closed conformation. Since closed syntaxin is unable to interact with its partner SNAREs SNAP-25 and synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc18–1 to syntaxin 1 with its biological function. We now show that in intact and exocytosis-competent lawns of plasma membrane, Munc18–1 forms a complex with syntaxin that allows formation of SNARE complexes. Munc18–1 associated with membrane-bound syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25–deficient mice are used. We conclude that Munc18–1 allows for the formation of a complex between syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis. [ABSTRACT FROM AUTHOR]

Membrane fusion in the secretory pathway is mediated by soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. Different fusion steps are thought to be effected by independent sets of SNAREs, but it is unclear whether specificity is determined by an intrinsic specificity of SNARE pairing or by upstream factors. Using a newly developed microscopy-based assay, we have investigated the SNARE specificity of homotypic early endosomal fusion. We show that early endosomes contain multiple sets of SNAREs, including, in addition to the putative early endosomal SNAREs, those involved in exocytosis and in fusion of late endosomes. We demonstrate that fusion is largely mediated by a complex formed by syntaxin 13, syntaxin 6, vti1a, and VAMP4, whereas the exocytic and late endosomal SNAREs play little or no role in the reaction. In contrast, proteoliposomes reconstituted with early endosomal SNAREs promiscuously fuse with liposomes containing exocytotic or late endosomal SNAREs. We conclude that the specificity of SNARE pairing does not suffice to determine the specificity of organelle fusion. [ABSTRACT FROM AUTHOR]

Tetraspanins emerge as a family of membrane proteins mediating an exceptional broad diversity of functions. The naming refers to their four transmembrane segments, which define the tetraspanins' typical membrane topology. In this study, we analyzed alternative splicing of tetraspanins. Besides isoforms with four transmembrane segments, most mRNA sequences are coding for isoforms with one, two or three transmembrane segments, representing structurally mono-, di- and trispanins. Moreover, alternative splicing may alter transmembrane topology, delete parts of the large extracellular loop, or generate alternative N- or C-termini. As a result, we define structure-based classes of non-conventional tetraspanins. The increase in gene products by alternative splicing is associated with an unexpected high structural variability of tetraspanins. We speculate that non-conventional tetraspanins have roles in regulating ER exit and modulating tetraspanin-enriched microdomain function. [ABSTRACT FROM AUTHOR]