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Epithelial folding mediated by apical constriction converts flat epithelial sheets into multilayered, complex tissue structures and is used throughout development in most animals (1). Little is known, however, about how forces produced near the apical surface of the tissue are transmitted within individual cells to generate the global changes in cell shape that characterize tissue deformation. Here we apply particle tracking velocimetry in gastrulating Drosophila embryos to measure the movement of cytoplasm and plasma membrane during ventral furrow formation (2,3). We find that cytoplasmic redistribution during the lengthening phase of ventral furrow formation can be precisely described by viscous flows that quantitatively match the predictions of hydrodynamics. Cell membranes move with the ambient cytoplasm, with little resistance to, or driving force on, the flow. Strikingly, apical constriction produces similar flow patterns in mutant embryos that fail to form cells before gastrulation ('aceUular' embryos), such that the global redistribution of cytoplasm mirrors the summed redistribution occurring in individual cells of wild-type embryos. Our results indicate that during the lengthening phase of ventral furrow formation, hydrodynamic behaviour of the cytoplasm provides the predominant mechanism transmitting apically generated forces deep into the tissue and that cell individualization is dispensable., During Drosophila gastrulation, ventrally localized prospective mesoderm forms a furrow and invaginates from the surface of the embryo. As this furrow forms, individual cells first constrict at their apical end [...]

Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier.sup.1 that defines cell shape.sup.2 and allows the cell to sustain large mechanical loads such as turgor pressure.sup.3. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope.sup.4,5. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.The outer membrane of Gram-negative bacteria is shown to be at least as stiff as the cell wall, and this property enables it to protect cells from mechanical pertubations., Author(s): Enrique R. Rojas [sup.1] [sup.2] [sup.3] , Gabriel Billings [sup.4] , Pascal D. Odermatt [sup.1] [sup.5] , George K. Auer [sup.6] , Lillian Zhu [sup.1] , Amanda Miguel [sup.1] [...]

Serine palmitoyltransferase (SPT) is the first and rate-limiting enzyme of the de novo biosynthetic pathway of sphingomyelin (SM). Both SPT and SM have been implicated in the pathogenesis of atherosclerosis, [...]

More than half of mammalian genes generate multiple messenger RNA isoforms that differ in their 3' untranslated regions (3' UTRs) and therefore in regulatory sequences (1), often associated with cell [...]

ARTICLE INFO Keywords: Anti-Candida Mono-Terpenoids Linalool Citral Citronellal Linalyl acetate Eugenol Benzyl benzoate ABSTRACT Anti-Candida potential of six terpenoids were evaluated in this study against various isolates of Candida albicans [...]

Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitors in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis. doi/ 10.1073/pnas.1007747108

Our previous studies of microperfused single proximal tubule showed that flow-dependent [Na.sup.+] and HC[O.sub.3.sup.-] reabsorption is due to a modulation of both NHE3 and vacuolar [H.sup.+]-ATPase (V-ATPase) activity. An intact actin cytoskeleton was indicated to provide a structural framework for proximal tubule cells to transmit mechanical forces and subsequently modulate cellular functions. In this study, we have used mouse proximal tubule (MPT) cells as a model to study the role of fluid shear stress (FSS) on apical NHE3 and V-ATPase and basolateral Na/K-ATPase trafficking and expression. Our hypothesis is that FSS stimulates both apical and basolateral transporter expression and trafficking, which subsequently mediates salt and volume reabsorption. We exposed MPT cells to 0.2 dynes/[cm.sup.2] FSS for 3 h and performed confocal microscopy and Western blot analysis to compare the localization and expression of both apical and basolateral transporters in control cells and cells subjected to FSS. Our findings show that FSS leads to an increment in the amount of protein expression, and a translocation of apical NHE3 and V-ATPase from the intracellular compartment to the apical plasma membrane and Na/K-ATPase to the basolateral membrane. Disrupting actin by cytochalasin D blocks the FSS-induced changes in NHE3 and Na/K-ATPase, but not V-ATPase. In contrast, FSS-induced V-ATPase redistribution and expression are largely inhibited by colchicine, an agent that blocks microtubule polymerization. Our findings suggest that the actin cytoskeleton plays an important role in FSS-induced NHE3 and Na/K-ATPase trafficking, and an intact microtubule network is critical in FSS-induced modulation of V-ATPase in proximal tubule cells. cell mechanotransduction | ion transporter | protein trafficking | glomerular tubular balance | epithelial cells doi: 10.1073/pnas.1015751107

The activity of P-type plasma membrane [H.sup.+]-ATPases is modulated by [H.sup.+] and cations, with [K.sup.+] and [Ca.sup.2+] being of physiological relevance. Using X-ray crystallography, we have located the binding site for [Rb.sup.+] as a [K.sup.+] congener, and for [Tb.sup.3+] and [Ho.sup.3+]as [Ca.sup.2+][ congeners. [Rb.sup.+] is found coordinated by a conserved aspartate residue in the phosphorylation domain. A single [Tb.sup.3+] ion is identified positioned in the nucleotide-binding domain in close vicinity to the bound nucleotide. [Ho.sup.3+] ions are coordinated at two distinct sites within the [H.sup.+]-ATPase: One site is at the interface of the nucleotide-binding and phosphorylation domains, and the other is in the transmembrane domain toward the extracellular side. The identified binding sites are suggested to represent binding pockets for regulatory cations and a [H.sup.+] binding site for protons leaving the pump molecule. This implicates [Ho.sup.3+] as a novel chemical tool for identification of proton binding sites. allosteric regulation | membrane protein crystallography | proton transport doi/ 10.1073/pnas.1010416107

Once excess liquid gains access to air spaces of an injured lung, the act of breathing creates and destroys foam and thereby contributes to the wounding of epithelial cells by interfacial stress. Since cells are not elastic continua, but rather complex network structures composed of solid as well as liquid elements, we hypothesize that plasma membrane (PM) wounding is preceded by a phase separation, which results in blebbing. We postulate that interventions such as a hypertonic treatment increase adhesive PM-cytoskeletal (CSK) interactions, thereby preventing blebbing as well as PM wounds. We formed PM tethers in alveolar epithelial cells and fibroblasts and measured their retractive force as readout of PM-CSK adhesive interactions using optical tweezers. A 50-mOsm increase in media osmolarity consistently increased the tether retractive force in epithelial cells but lowered it in fibroblasts. The osmo-response was abolished by pretreatment with latrunculin, cytochalasin D, and calcium chelation. Epithelial cells and fibroblasts were exposed to interfacial stress in a microchannel, and the fraction of wounded cells were measured. Interventions that increased PM-CSK adhesive interactions prevented blebbing and were cytoprotective regardless of cell type. Finally, we exposed ex vivo perfused rat lungs to injurious mechanical ventilation and showed that hypertonic conditioning reduced the number of wounded subpleural alveolus resident cells to baseline levels. Our observations support the hypothesis that PM-CSK adhesive interactions are important determinants of the cellular response to deforming stress and pave the way for preclinical efficacy trials of hypertonic treatment in experimental models of acute lung injury. alveolar epithelial cell; acute lung injury; interfacial stress; osmotic response; cytoprotection doi: 10.1152/ajplung.00217.2010.

Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis. molecular docking | dynamic instability | nanodomains | total internal reflection fluorescence doi/ 10.1073/pnas.1014823107

Microtubules (MTs) contribute to key processes during cell motility, including the regulation of focal adhesion turnover and the establishment and maintenance of cell orientation. It was previously demonstrated that the ErbB2 receptor tyrosine kinase regulated MT outgrowth to the cell cortex via a complex including Memo, the GTPase RhoA, and the formin mDial. But the mechanism that linked this signaling module to MTs remained undefined. We report that ErbB2-induced repression of glycogen synthase kinase-3 (GSK3) activity, mediated by Memo and mDial, is required for MT capture and stabilization. Memo-dependent inhibition of GSK3 allows the relocalization of APC (adenomatous polyposis coil) and cytoplasmic linker-associated protein 2 (CLASP2), known MT-associated proteins, to the plasma membrane and ruffles. Peripheral microtubule extension also requires expression of the plus-end binding protein EB1 and its recently described interactor, the spectraplakin ACF7. In fact, in migrating cells, ACF7 localizes to the plasma membrane and ruffles, in a Memo-, GSK3-, and APC-dependent manner. Finally, we demonstrate that ACF7 targeting to the plasma membrane is both required and sufficient for MT capture downstream of ErbB2. This function of ACF7 does not require its recently described ATPase activity. By defining the signaling pathway by which ErbB2 allows MT capture and stabilization at the cell leading edge, we provide insights into the mechanism underlying cell motility and steering. cell motility | receptor tyrosine kinase | Memo protein | glycogen synthase kinase-3 | adenomatous polyposis coli doi/ 10.1073/pnas.1000975107

Exocytosis requires formation of SNARE [soluble N-ethylmaleimide--sensitive factor attachment protein (SNAP) receptor] complexes between vesicle and target membranes. Recent assessments in reduced model systems have produced divergent estimates of the number of SNARE complexes needed for fusion. Here, we used a titration approach to answer this question in intact, cultured chromaffin cells. Simultaneous expression of wild-type SNAP-25 and a mutant unable to support exocytosis progressively altered fusion kinetics and fusion-pore opening, indicating that both proteins assemble into heteromeric fusion complexes. Expressing different wild-type:mutant ratios revealed a third-power relation for fast (synchronous) fusion and a near-linear relation for overall release. Thus, fast fusion typically observed in synapses and neurosecretory cells requires at least three functional SNARE complexes, whereas slower release might occur with fewer complexes. Heterogeneity in SNARE-complex number may explain heterogeneity in vesicular release probability. 10.1126/science.1193134

Antiviral drugs targeting viral proteins often result in prompt selection for resistance. Moreover, the number of viral targets is limited. Novel antiviral targets are therefore needed. The unique characteristics of fusion between virion envelopes and cell membranes may provide such targets. Like all fusing bilayers, viral envelopes locally adopt hourglass-shaped stalks during the initial stages of fusion, a process that requires local negative membrane curvature. Unlike cellular vesicles, however, viral envelopes do not redistribute lipids between leaflets, can only use the energy released by virion proteins, and fuse to the extracellular leaflets of cell membranes. Enrichment in phospholipids with hydrophilic heads larger than their hydrophobic tails in the convex outer leaflet of vesicles favors positive curvature, therefore increasing the activation energy barrier for fusion. Such phospholipids can increase the activation barrier beyond the energy provided by virion proteins, thereby inhibiting viral fusion. However, phospholipids are not pharmacologically useful. We show here that a family of synthetic rigid amphiphiles of shape similar to such phospholipids, RAFIs (rigid amphipathic fusion inhibitors), inhibit the infectivity of several otherwise unrelated enveloped viruses, including hepatitis C and HSV-1 and -2 (lowest apparent IC50 48 nM), with no cytotoxic or cytostatic effects (selectivity index > 3,000) by inhibiting the increased negative curvature required for the initial stages of fusion. lipid bilayer fusion | DNA virus | RNA virus | Sindbis virus | nucleosides doi/ 10.1073/pnas.1010026107

Microglia, the immune cells of the central nervous system, are attracted to sites of injury. The injury releases adenosine triphosphate (ATP) into the extracellular space, activating the microglia, but the full mechanism of release is not known. In glial cells, a family of physiologically regulated unpaired gap junction channels called innexons (invertebrates) or pannexons (vertebrates) located in the cell membrane is permeable to ATE Innexons, but not pannexons, also pair to make gap junctions. Glial calcium waves, triggered by injury or mechanical stimulation, open pannexon/innexon channels and cause the release of ATE It has been hypothesized that a glial calcium wave that triggers the release of ATP causes rapid microglial migration to distant lesions. In the present study in the leech, in which a single giant glial cell ensheathes each connective, hydrolysis of ATP with 10 U/ml apyrase or block of innexons with 10 taM carbenoxolone (CBX), which decreased injury-induced ATP release, reduced both movement of microglia and their accumulation at lesions. Directed movement and accumulation were restored in CBX by adding ATP, consistent with separate actions of ATP and nitric oxide, which is required for directed movement but does not activate glia. Injection of glia with innexin2 (Hminx2) RNAi inhibited release of carboxyfluorescein dye and microglial migration, whereas injection of innexin1 (Hminx1) RNAi did not when measured 2 days after injection, indicating that glial cells' ATP release through innexons was required for microglial migration after nerve injury. Focal stimulation either mechanically or with ATP generated a calcium wave in the glial cell; injury caused a large, persistent intracellular calcium response. Neither the calcium wave nor the persistent response required ATP or its release. Thus, in the leech, innexin membrane channels releasing ATP from glia are required for migration and accumulation of microglia after nerve injury. doi/ 10.1085/jgp.201010476

During response of budding yeast to peptide mating pheromone, the cell becomes markedly polarized and MAPK scaffold protein Ste5 localizes to the resulting projection (shmoo tip). We demonstrated before that this recruitment is essential for sustained MAPK signaling and requires interaction of a pleckstrin homology (PH) domain in Ste5 with phosphatidylinositol 4,5-bisphosphate [PtdIns (4,5)[P.sub.2]] in the plasma membrane. Using fluorescently tagged high-affinity probes specific for Ptdlns(4,5)[P.sub.2], we have now found that this phosphoinositide is highly concentrated at the shmoo tip in cells responding to pheromone. Maintenance of this strikingly anisotropic distribution of PtdIns(4,5)[P.sub.2], stable tethering of Ste5 at the shmoo tip, downstream MAPK activation, and expression of a mating pathway-specific reporter gene all require continuous function of the plasma membrane-associated PtdIns 4-kinase Stt4 and the plasma membrane-associated Ptdlns4P 5-kinase Mss4 (but not the Golgi-associated PtdIns 4-kinase Pik1). Our observations demonstrate that PtdIns(4,5)[P.sub.2] is the primary determinant for restricting localization of Ste5 within the plasma membrane and provide direct evidence that an extracellular stimulus-evoked self-reinforcing mechanism generates a spatially enriched pool of PtdIns (4,5)[P.sub.2] necessary for the membrane anchoring and function of a signaling complex. [alpha]-factor | phosphoinositides | scaffold protein I yeast mating response doi/ 10.1073/pnas.1005817107

The heptameric coatomer complex forms the protein shell of membrane-bound vesicles that are involved in transport from the Golgi to the endoplasmatic reticulum and in intraGolgi trafficking. The heptamer can be dissected into a heterotetrameric F-subcomplex, which displays similarities to the adapter complex of the 'inner' coat in clathrin-coated vesicles, and a heterotrimeric B-subcomplex, which is believed to form an 'outer' coat with a morphology distinct from that of clathrin-coated vesicles. We have determined the crystal structure of the complex between the c-terminal domain (CTD) of [alpha]-COP and full-length c-COP, two components of the B-subcomplex, at a 2.9 [Angstrom] resolution. The [alpha]-[COP.sup.CTD] * c-COP heterodimer forms a rod-shaped structure, in which c-COP adopts a tetratricopeptide repeat (TPR) fold that deviates substantially from the canonical superhelical conformation. The [alpha]-COP CTD adopts a U-shaped architecture that complements the TPR fold of c-COR The c-COP TPRs form a circular bracelet that wraps around a protruding [beta]-hairpin of the [alpha]-COP CTD, thus interlocking the two proteins. The [alpha]-[COP.sup.CTD] * c-COP complex forms heterodimers in solution, and we demonstrate biochemically that the heterodimer directly interacts with the Dsll tethering complex. These data suggest that the heterodimer is exposed on COPI vesicles, while the remaining part of the B-subcomplex oligomerizes underneath into a cage. coatomer | interaction studies | membrane coat | U-shaped [alpha]-helical solenoid | tetratricopeptide repeat (TPR) doi/ 10.1073/pnas.1006297107

The delivery of AMPA receptors to the plasma membrane is a critical step both for the synaptic delivery of these receptors and for the regulation of synaptic transmission. To directly visualize fusion events of transport vesicles containing the AMPA receptor GluA2 subunit with the plasma membrane we used pHluorin-tagged GluA2 subunits and total internal reflection fluorescence microscopy. We demonstrate that the plasma membrane insertion of GluA2 requires the NSF binding site within its intracellular cytoplasmic domain and that RNA editing of the Q/R site in the ion channel region plays a key role in GluA2 plasma membrane insertion. Finally, we show that plasma membrane insertion of heteromeric GluA2/3 receptors follows the same rules as homomeric GluA2 receptors. These results demonstrate that the plasma membrane delivery of GluA2 containing AMPA receptors is regulated by its unique structural elements. glutamate receptors | membrane trafficking | synapse | synaptic plasticity doi/ 10.1073/pnas.1006584107

Rapid plasma membrane resealing is essential for cellular survival. Earlier studies showed that plasma membrane repair requires [Ca.sup.2+]-dependent exocytosis of lysosomes and a rapid form of endocytosis that removes membrane lesions. However, the functional relationship between lysosomal exocytosis and the rapid endocytosis that follows membrane injury is unknown. In this study, we show that the lysosomal enzyme acid sphingomyelinase (ASM) is released extracellularly when cells are wounded in the presence of [Ca.sup.2+]. ASM-deficient cells, including human cells from Niemann-Pick type A (NPA) patients, undergo lysosomal exocytosis after wounding but are defective in injury-dependent endocytosis and plasma membrane repair. Exogenously added recombinant human ASM restores endocytosis and resealing in ASM-depleted cells, suggesting that conversion of plasma membrane sphingomyelin to ceramide by this lysosomal enzyme promotes lesion internalization. These findings reveal a molecular mechanism for restoration of plasma membrane integrity through exocytosis of lysosomes and identify defective plasma membrane repair as a possible component of the severe pathology observed in NPA patients. doi/10.1083/jcb.201003053

Cellular organelle membranes maintain their integrity, global shape, and composition despite vigorous exchange among compartments of lipids and proteins during trafficking and signaling. Organelle homeostasis involves dynamic molecular sorting mechanisms that are far from being understood. In contrast, equilibrium thermodynamics of membrane mixing and sorting, particularly the phase behavior of binary and ternary model membrane mixtures and its coupling to membrane mechanics, is relatively well characterized. Elucidating the continuous turnover of live cell membranes, however, calls for experimental and theoretical membrane models enabling manipulation and investigation of directional mass transport. Here we introduce the phenomenon of curvature-induced domain nucleation and growth in membrane mixtures with fluid phase coexistence. Membrane domains were consistently observed to nucleate precisely at the junction between a strongly curved cylindrical (tube) membrane and a pipette-aspirated giant unilamellar vesicle. This experimental geometry mimics intracellular sorting compartments, because they often show tubular-vesicular membrane regions. Nucleated domains at tube necks were observed to present diffusion barriers to the transport of lipids and proteins. We find that curvature-nucleated domains grow with characteristic parabolic time dependence that is strongly curvature-dependent. We derive an analytical model that reflects the observed growth dynamics. Numerically calculated membrane shapes furthermore allow us to elucidate mechanical details underlying curvaturedependent directed lipid transport. Our observations suggest a novel dynamic membrane sorting principle that may contribute to intracellular protein and lipid sorting and trafficking. liquid disordered | liquid ordered | phase transition | vesicle doi/ 10.1073/pnas.0913997107

Precisely controlled growth and morphogenesis of developing epithelial tissues require coordination of multiple factors, including proliferation, adhesion, cell shape, and apoptosis. RhoA, a small GTPase, is known to control epithelial morphogenesis and integrity through its ability to regulate the cytoskeleton. In this study, we examine a less well-characterized RhoA function in cell survival. We demonstrate that the Drosophila melanogaster RhoA, Rho1, promotes apoptosis independently of Rho kinase through its effects on c-Jun N[H.sub.2]-terminal kinase (JNK) signaling. In addition, Rho1 forms a complex with Slipper (Slpr), an upstream activator of the JNK pathway. Loss of Moesin (Moe), an upstream regulator of Rho1 activity, results in increased levels of Rho1 at the plasma membrane and cortical accumulation of Slpr. Together, these results suggest that Rho1 functions at the cell cortex to regulate JNK activity and implicate Rho1 and Moe in epithelial cell survival. doi/10.1083/jcb.200912010

We describe an optical method capable of tracking a single fluorescent molecule with a flexible choice of high spatial accuracy (~10-20 nm standard deviation or ~20-40 nm full-width-at-half-maximum) and temporal resolution ( single molecule | tracking | membrane dynamics | lipid rafts doi/ 10.1073/pnas.0912894107

Primary cilia are microtubule-based membrane projections located at the surface of many cells. Defects in primary cilia formation have been implicated in a number of genetic disorders, such as Bardet-Biedl Syndrome and Polycystic Kidney Disease. Recent studies have demonstrated that polarized vesicular transport involving Rab8 and its guanine nucleotide-exchange factor Rabin8 is essential for primary ciliogenesis. Here we report that Rabin8 is a direct downstream effector of Rab11, which functions in membrane trafficking from the trans-Golgi network and recycling endosomes. Rab11, in its GTP-bound form, interacts with Rabin8 and kinetically stimulates the guanine nucleotide-exchange activity of Rabin8 toward Rab8. Rab11 is enriched at the base of the primary cilia and inhibition of Rab11 function by a dominant-negative mutant or RNA interference blocks primary ciliogenesis. Our results suggest that Rab GTPases coordinate with each other in the regulation of vesicular trafficking during primary ciliogenesis. primary cilia | Rabin8 | recycling endosome | BBS | exocyst doi/10.73/pnas.1002401107

Uncovering the plasma membrane distribution of tyrosine kinase Lck is crucial to understanding T lymphocyte triggering. Several studies of Lck species partitioning have given contradictory results. We decided to re-address this point by using phospho-specific antibodies to characterize active and inactive Lck partitioning in raft and non-raft membranes from primary human peripheral blood T lymphocytes. We show that most inactive Lck was localized in rafts and was associated with nearly all CD4 coreceptors and its negative regulator Csk in resting cells, while T cell receptor (TCR) engagement promoted a sustained dephosphorylation of inactive Lck. In contrast, active Lck had a more discrete distribution interacting with only a small number of CD4 coreceptors, and the kinase showed a rapid and short phosphorylation after TCR triggering. The differences in distribution and kinetics may be related to T lymphocyte signalling threshold modulation by Lck species and suggest how TCR triggering is first initiated. This study furthers our knowledge of the TCR activation model in primary human T lymphocytes. Key words: protein kinases, T cell receptor, protein phosphatases, lipid raft, leukocyte-specific protein tyrosine kinase, coreceptor CD4, human. La mise en evidence de la distribution de la tyrosine kinase Lck dans la membrane plasmique est essentielle pour comprendre l'activation des lymphocytes T. Malgre de nombreuses etudes sur le sujet, les resultats sur la repartition de Lck demeurent contradictoires. Nous avons decide de reexaminer cette question et de caracteriser plus en detail, a l'aide d'anticorps phosphospecifiques, la repartition des Lck actives et inactives dans les radeaux lipidiques membranaires et dans les membranes sans radeau de lymphocytes T primaires circulants humains. Nous montrons que la Lck la plus inactive se situe dans les radeaux et qu'elle est associeea presque tous les corecepteurs CD4 et a son regulateur neegatif Csk dans les cellules au repos, alors que l'engagement du RCT favorise une deephosphorylation soutenue de la Lck negative. A l'oppose, la Lck active a une distribution plus discrete, interagissant seulement avec un petit nombre de CD4, et montre une phosphorylation rapide et breve apres l'activation du RCT. Les differences dans la distribution et la cinetique pourraient etre liees a la modulation du seuil de signalisation des lymphocytes T par Lck, et a la maniere dont l'activation du RCT est deeclencheee, ce qui ameliore notre comprehension du modele d'activation du RCT dans un modele de lymphocytes T primaires humains. Mots-cles: proteines kinases, recepteur des cellules T, proteines phosphatases, radeau lipidique, proteine tyrosine kinase specifique des leucocytes, corecepteur CD4, humain. [Traduit par la Redaction], Introduction Communication occurs between T lymphocytes and antigen-presenting cells by a well-organized and dynamic T cell receptor (TCR) signalling machinery. Phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) in the [...]

We present the first atomic-resolution observations of permeation and gating in a [K.sup.+] channel, based on molecular dynamics simulations of the Kv1.2 pore domain. Analysis of hundreds of simulated permeation events revealed a detailed conduction mechanism, resembling the Hodgkin--Keynes 'knock-on' model, in which translocation of two selectivity filter--bound ions is driven by a third ion; formation of this knock-on intermediate is rate determining. In addition, at reverse or zero voltages, we observed pore closure by a novel 'hydrophobic gating' mechanism: A dewetting transition of the hydrophobic pore cavity--fastest when [K.sup.+] was not bound in selectivity filter sites nearest the cavity--caused the open, conducting pore to collapse into a closed, nonconducting conformation. Such pore closure corroborates the idea that voltage sensors can act to prevent pore collapse into the intrinsically more stable, closed conformation, and it further suggests that molecular-scale dewetting facilitates a specific biological function: [K.sup.+] channel gating. Existing experimental data support our hypothesis that hydrophobic gating may be a fundamental principle underlying the gating of voltage-sensitive [K.sup.+] channels. We suggest that hydrophobic gating explains, in part, why diverse ion channels conserve hydrophobic pore cavities, and we speculate that modulation of cavity hydration could enable structural determination of both open and closed channels. ion channel | ion permeation | membrane | electrophysiology | dewetting doi/10.1073/pnas.0911691107

The capabilities of time-of-flight secondary ion mass spectrometry (TOF-SIMS) with regards to limits in lateral resolution for biological samples are examined using supported lipid bilayers and individual lipid vesicles, both being among the most commonly used cell membrane mimics. Using supported 1-palmitoyl-2-oleoyl-sn-giycero3-phosphocholine (POPC) bilayers confined to a Si[O.sub.2] substrate by a chemically modified gold surface, the edge of the lipid bilayer was analyzed by imaging TOF-SIMS to assess the lateral resolution. The results using 80 keV [Bi.sub.3.sup.2] primary ions show that, under optimized conditions, mass spectrometry imaging of specific unlabeled lipid fragments is possible with sub-100 nm lateral resolution. Comparison of the secondary ion yields for the phosphocholine ion (m/z 184) from a POPC bilayer using [C.sub.60] or [Bi.sub.3] primary ions showed similar results, indicating an advantage of [Bi.sub.3] primary ions for high-resolution imaging of lipid membranes, due to their better demonstrated focusing capability. Moreover, using 300 nm vesicles of different lipid composition, the capability to detect and chemically identify individual submicrometer lipid vesicles at separations down to ~1 [micro]m is demonstrated. 10.1021/ac902744u

The recognition and binding of cholesterol is an important feature of many eukaryotic, viral, and prokaryotic proteins, but the molecular details of such interactions are understood only for a few proteins. The pore-forming cholesterol-dependent cytolysins (CDCs) contribute to the pathogenic mechanisms of a large number of Gram-positive bacteria. Cholesterol dependence of the CDC mechanism is a hallmark of these toxins, yet the identity of the CDC cholesterol recognition motif has remained elusive. A detailed analysis of membrane interactive structures at the tip of perfringolysin O (PFO) domain 4 reveals that a threonine-leucine pair mediates CDC recognition of and binding to membrane cholesterol. This motif is conserved in all known CDCs and conservative changes in its sequence or order are not well tolerated. Thus, the Thr-Leu pair constitutes a common structural basis for mediating CDC-cholesterol recognition and binding, and defines a unique paradigm for membrane cholesterol recognition by surface-binding proteins. hemolysin | intermedilysin | sterol | toxin | pore doi/10.1073/pnas.0911581107

The intestinal epithelium serves as a major protective barrier between the mammalian host and the external environment. Here we show that the transmembrane serine protease matriptase plays a pivotol role in the formation and integrity of the intestinal epithelial barrier. St14 hypomorphic mice, which have a 100-fold reduction in intestinal matriptase mRNA levels, display a 35% reduction in intestinal transepithelial electrical resistance (TEER). Matriptase is expressed during intestinal epithelial differentiation and colocalizes with E-cadherin to apical junctional complexes (AJC) in differentiated polarized Caco-2 monolayers. Inhibition of matriptase activity using a specific peptide inhibitor or by knockdown of matriptase by siRNA disrupts the development of TEER in barrier-forming Caco-2 monolayers and increases paracellular permeability to macromolecular FITC-dextran. Loss of matriptase was associated with enhanced expression and incorporation of the permeability-associated, 'leaky' tight junction protein claudin-2 at intercellular junctions. Knockdown of claudin-2 enhanced the development of TEER in matriptase-silenced Caco-2 monolayers, suggesting that the reduced barrier integrity was caused, at least in part, by an inability to regulate claudin-2 expression and incorporation into junctions. We find that matriptase enhances the rate of claudin-2 protein turnover, and that this is mediated indirectly through an atypical PKC[zeta]-dependent signaling pathway. These results support a key role for matriptase in regulating intestinal epithelial barrier competence, and suggest an intriguing link between pericellular serine protease activity and tight junction assembly in polarized epithelia. claudin-2 | intestinal barrier | St14 | type II transmembrane serine protease | tight junction doi/10.1073/pnas.0903923107

The generation of membrane curvature in intracellular traffic involves many proteins that can curve lipid bilayers. Among these, dynamin-like proteins were shown to deform membranes into tubules, and thus far are the only proteins known to mechanically drive membrane fission. Because dynamin forms a helical coat circling a membrane tubule, its polymerization is thought to be responsible for this membrane deformation. Here we show that the force generated by dynamin polymerization, 18 pN, is sufficient to deform membranes yet can still be counteracted by high membrane tension. Importantly, we observe that at low dynamin concentration, polymer nucleation strongly depends on membrane curvature. This suggests that dynamin may be precisely recruited to membrane buds' necks because of their high curvature. To understand this curvature dependence, we developed a theory based on the competition between dynamin polymerization and membrane mechanical deformation. This curvature control of dynamin polymerization is predicted for a specific range of concentrations (~0.1-10 [micro]M), which corresponds to our measurements. More generally, we expect that any protein that binds or self-assembles onto membranes in a curvature-coupled way should behave in a qualitatively similar manner, but with its own specific range of concentration. force | nucleation | fission | endocytosis | dynamin-like proteins doi/10.1073/pnas.0913734107

ATP synthase catalyzes ATP synthesis at the expense of an electrochemical ion gradient across a membrane that can be generated by different exergonic reactions. Sulfur reduction is the main energy-yielding reaction in the hyperthermophilic strictly anaerobic Crenarchaeon Ignicoccus hospitalis. This organism is unusual in having an inner and an outer membrane that are separated by a huge intermembrane compartment. Here we show, on the basis of immuno-EM analyses of ultrathin sections and immunofluorescence experiments with whole I. hospitalis cells, that the ATP synthase and [H.sub.2]:sulfur oxidoreductase complexes of this organism are located in the outer membrane. These two enzyme complexes are mandatory for the generation of an electrochemical gradient and for ATP synthesis. Thus, among all prokaryotes possessing two membranes in their cell envelope (including Planctomycetes, Gram-negative bacteria), I. hospitalis is a unique organism, with an energized outer membrane and ATP synthesis within the periplasmic space. In addition, DAPI staining and EM analyses showed that DNA and ribosomes are localized in the cytoplasm, leading to the conclusion that in I. hospitalis energy conservation is separated from information processing and protein biosynthesis. This raises questions regarding the function of the two membranes, the interaction between these compartments, and the general definition of a cytoplasmic membrane. Archaea | ATP synthase | ATPase | immunolabeling | sulfur reductase www.pnas.org/cgi/doi/10.1073/pnas.0911711107

Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane--cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the post-fusion granule membrane--plasma membrane complex. Experiments on dil-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin. doi/10.1083/jcb.200908010

The anticancer peptide PNC-27, which contains an HDM-2-binding domain corresponding to residues 12-26 of p53 and a transmembrane-penetrating domain, has been found to kill cancer cells (but not normal cells) by inducing membranolysis. We find that our previously determined 3D structure of the p53 residues of PNC-27 is directly superimposable on the structure for the same residues bound to HDM-2, suggesting that the peptide may target HDM-2 in the membranes of cancer cells. We now find significant levels of HDM-2 in the membranes of a variety of cancer cells but not in the membranes of several untransformed cell lines. In colocalization experiments, we find that PNC-27 binds to cell membrane-bound HDM-2. We further transfected a plasmid expressing full-length HDM-2 with a membrane-localization signal into untransformed MCF-10-2A cells not susceptible to PNC-27 and found that these cells expressing full-length HDM-2 on their cell surface became susceptible to PNC-27. We conclude that PNC-27 targets HDM-2 in the membranes of cancer cells, allowing it to induce membranolysis of these cells selectively. HDM-2 binding | membranolysis | three-dimensional structure | transfection doi/10.1073/pnas.0909364107

Structural analyses of the extracellular region of stem cell factor (SCF) receptor (also designated KIT) in complex with SCF revealed a sequence motif in a loop in the fourth Ig-like domain (D4) that is responsible for forming homotypic receptor contacts and for ligand-induced KIT activation and cell signaling. An identical motif was identified in the most membrane-proximal seventh Ig-like domain (D7) of vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and VEGFR3. In this report we demonstrate that ligand-induced tyrosine autophosphorylation and cell signaling via VEGFR1 or VEGFR2 harboring mutations in critical residues (Arg726 or Asp731) in D7 are strongly impaired. We also describe the crystal structure of D7 of VEGFR2 to a resolution of 2.7 [Angstrom]. The structure shows that homotypic D7 contacts are mediated by salt bridges and van der Waals contacts formed between Arg726 of one protomer and Asp731 of the other protomer. The structure of D7 dimer is very similar to the structure of D4 dimers seen in the crystal structure of KIT extracellular region in complex with SCF. The high similarity between VEGFR D7 and KIT D4 in both structure and function provides further evidence for common ancestral origins of type III and type V RTKs. It also reveals a conserved mechanism for RTK activation and a novel target for pharmacological intervention of pathologically activated RTKs. angiogenesis | cancer | phosphorylation | protein kinases | surface receptors doi/10.1073/pnas.0914052107

The [Na.sup.+]/[K.sup.+]-ATPase mediates electrogenic transport by exporting three [Na.sup.+] ions in exchange for two [K.sup.+] ions across the cell membrane per adenosine triphosphate molecule. The location of two [Rb.sup.+] ions in the crystal structures of the [Na.sup.+]/[K.sup.+]-ATPase has defined two 'common' cation binding sites, I and II, which accommodate [Na.sup.+] or [K.sup.+] ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this 'unique' [Na.sup.+] binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human [Na.sup.+]/[K.sup.+]-ATPase [[alpha].sub.2] subunit decreases the affinity for extracellular and intracellular [Na.sup.+], in agreement with previous biochemical studies. Apparently, the [DELTA]YY deletion changes [Na.sup.+] affinity at site III but leaves the common sites unaffected, whereas the more extensive [DELTA]KETYY deletion affects the unique site and the common sites as well. In the absence of extracellular [K.sup.+], the [DELTA]YY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were [Na.sup.+] dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under [Na.sup.+]/[Na.sup.+] exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular [Na.sup.+] release/binding mechanism. In analogy to the mechanism proposed for the [H.sup.+] leak currents of the wild-type [Na.sup.+]/[K.sup.+]-ATPase, we suggest that the [DELTA]YY deletion lowers the energy barrier for the intracellular [Na.sup.+] occlusion reaction, thus destabilizing the [Na.sup.+]-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the [Na.sup.+]/[K.sup.+]-ATPase [alpha] subunit represents a structural and functional relay between [Na.sup.+] binding site III and the intracellular cation occlusion gate. doi/ 10.1085/jgp.200910301

Membrane binding of Gag, a crucial step in HIV-1 assembly, is facilitated by bipartite signals within the matrix (MA) domain: N-terminal myristoyl moiety and the highly basic region (HBR). We and others have shown that Gag interacts with a plasmamembrane-specific acidic phospholipid, phosphatidylinositol(4,5)-bisphosphate [PI(4,5)P2], via the HBR, and that this interaction is important for efficient membrane binding and plasma membrane targeting of Gag. Generally, in protein-Pl(4,5)[P.sub.2] interactions, basic residues promote the interaction as docking sites for the acidic headgroup of the lipid. In this study, toward better understanding of the Gag-PI(4,5)[P.sub.2] interaction, we sought to determine the roles played by all of the basic residues in the HBR. We identified three basic residues promoting PI(4,5)[P.sub.2]-dependent Gag-membrane binding. Unexpectedly, two other HBR residues, Lys25 and Lys26, suppress membrane binding in the absence of PI(4,5)[P.sup.2] and prevent promiscuous intracellular localization of Gag. This inhibition of nonspecific membrane binding is likely through suppression of myristate-dependent hydrophobic interaction because mutating Lys25 and Lys26 enhances binding of Gag with neutral-charged liposomes. These residues were reported to bind RNA. Importantly, we found that RNA also negatively regulates Gag membrane binding. In the absence but not presence of PI(4,5)[P.sub.2], RNA bound to MA HBR abolishes Gagliposome binding. Altogether, these data indicate that the HBR is unique among basic phosphoinositide-binding domains, because it integrates three regulatory components, PI(4,5)[P.sub.2], myristate, and RNA, to ensure plasma membrane specificity for particle assembly. basic residues | PI(4,5)[P.sub.2] | phosphoinositide | plasma membrane | retrovirus assembly doi/ 10.1073/pnas.0908661107

The broadly neutralizing anti-HIV antibody 4E10 recognizes an epitope very close to the virus membrane on the glycoprotein gp41. It was previously shown that epitope recognition improves in a membrane context and that 4E10 binds directly, albeit weakly, to lipids. Furthermore, a crystal structure of Fab 4E10 complexed to an epitope peptide revealed that the centrally placed, protruding H3 loop of the antibody heavy chain does not form peptide contacts. To investigate the hypothesis that the H3 loop apex might interact with the viral membrane, two Trp residues in this region were substituted separately or in combination with either Ala or Asp by site-directed mutagenesis. The resultant IgG variants exhibited similar affinities for an epitope peptide as WT 4E10 but lower apparent affinities for both viral membrane mimetic liposomes and Env(-) virus. Variants also exhibited lower apparent affinities for Env(+) virions and failed to significantly neutralize a number of 4E10-sensitive viruses. For the extremely sensitive HXB2 virus, variants did neutralize, but at 37- to >250-fold lower titers than WT 4E10, with Asp substitutions exerting a greater effect on neutralization potency than Ala substitutions. Because reductions in lipid binding reflect trends in neutralization potency, we conclude that Trp residues in the antibody H3 loop enable membrane proximal epitope recognition through favorable lipid interactions. The requirement for lipophilic residues such as Trp adjacent to the antigen binding site may explain difficulties in eliciting 4E10-1ike neutralizing antibody responses by immunization and helps define a unique motif for antibody recognition of membrane proximal antigens. 4E10 | gp41 | HIV I neutralizing antibody | tryptophan doi/ 10.1073/pnas.0909680107

There is now a significant interest in being able to locate single cells within geometrically defined regions of a microfluidic chip and to gain intracellular access through the local electroporation of the cell membrane. This paper describes the microfabricafion of electroporation devices which can enable the regional electroporation of adult ventricular myocytes, in order to lower the local electrical resistance of the cell membrane. Initially three different devices, designed to suit the characteristic geometry of the cardiomyocyte, were investigated (all three designs serve to focus the electric field to selected regions of the cell). We demonstrate that one of these three devices revealed the sequence of cellular responses to field strengths of increasing magnitudes, namely, cell contraction, hypercontraction, and lysis. This same device required a reduced threshold voltage for each of these events, including in particular membrane breakdown. We were not only able to show the gradual regional increase in the electric conductivity of the cell membrane but were also able to avoid changes in the local intra- and extracellular pH (by preventing the local generation of protons at the electrode surface, as a consequence of the reduced threshold voltage). The paper provides evidence for new slyategies for achieving robust and reproducible regional electroporation, a technique which, in future, may be used for the insertion of large molecular weight molecules (including genes) as well as for on-chip voltage clamping of the primary adult cardiomyocyte. 10.1021/ac901886j

Cell membranes display a tremendous complexity of lipids and proteins designed to perform the functions cells require. To coordinate these functions, the membrane is able to laterally segregate its constituents. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. Lipid rafts are fluctuating nanoscale assemblies of sphingolipid, cholesterol, and proteins that can be stabilized to coalesce, forming platforms that function in membrane signaling and trafficking. Here we review the evidence for how this principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to selectively focus membrane bioactivity. 10.1126/science.1174621

The Golgi-associated four-phosphate adaptor protein 2 (FAPP2) has been shown to possess transfer activity for glucosylceramide both in vitro and in cells. We have previously shown that FAPP2 is involved in apical transport from the Golgi complex in epithelial MDCK cells. In this paper we assign an unknown activity for the protein as well as providing structural insight into protein assembly and a low-resolution envelope structure. By applying analytical ultracentrifugation and small-angle x-ray scattering, we show that FAPP2 is a dimeric protein in solution, having a curved shape 30 nm in length. The purified FAPP2 protein has the capability to form tubules from membrane sheets in vitro. This activity is dependent on the phosphoinositide-binding activity of the PH domain of FAPP2. These data suggest that FAPP2 functions directly in the formation of apical carriers in the trans-Golgi network. membrane tubulation | PH domain | phosphatidylinositol 4-phosphate | trans-Golgi network | small-angle x-ray scattering (SAXS) doi/10.1073/pnas.0911789106

Tail-anchored (TA) membrane proteins are involved in a variety of important cellular functions, including membrane fusion, protein translocation, and apoptosis. The ATPase Get3 (Asnal, TRC40) was identified recently as the endoplasmic reticulum targeting factor of TA proteins. Get3 consists of an ATPase and [alpha]-helical subdomain enriched in methionine and glycine residues. We present structural and biochemical analyses of Get3 alone as well as in complex with a TA protein, ribosome-associated membrane protein 4 (Ramp4). The ATPase domains form an extensive dimer interface that encloses 2 nucleotides in a head-to-head orientation and a zinc ion. Amide proton exchange mass spectrometry shows that the [alpha]-helical subdomain of Get3 displays considerable flexibility in solution and maps the TA protein-binding site to the [alpha]-helical subdomain. The non-hydrolyzable ATP analogue AMPPNP-[Mg.sup.2+]-and ADP-[Mg.sup.2+]-bound crystal structures representing the pre-and posthydrolysis states are both in a closed form. In the absence of a TA protein cargo, ATP hydrolysis does not seem to be possible. Comparison with the ADP x [AlF.sub.4.sup.-]-bound structure representing the transition state (Mateja A, et al. (2009) Nature 461:361-366) indicates how the presence of a TA protein is communicated to the ATP-binding site. In vitro membrane insertion studies show that recombinant Get3 inserts Ramp4 in a nucleotide-and receptor-dependent manner. Although ATP hydrolysis is not required for Ramp4 insertion per se, it seems to be required for efficient insertion. We postulate that ATP hydrolysis is needed to release Get3 from its receptor. Taken together, our results provide mechanistic insights into posttranslational targeting of TA membrane proteins by Get3. ATP-binding protein | crystal structure | Asnal/Trc40 | Get pathway | posttranslational targeting doi/10.1073/pnas.0910223106

The ability of carcinoma cells arising at primary sites to cross their underlying basement membrane (BM), a specialized form of extracellular matrix that subtends all epithelial cells, and to access the host vasculature are central features of the malignant phenotype. The initiation of the invasive phenotype has been linked to the aberrant expression of zinc-finger transcriptional repressors, like Snail1, which act by triggering an epithelial-mesenchymal cell-like transformation (EMT-like) via the regulation of largely undefined, downstream effectors. Herein, we find that Snail1 induces cancer cells to (i) degrade and perforate BM barriers, (ii) initiate angiogenesis, and (iii) and intravasate vascular networks in vivo via a matrix metalloproteinase (MMP)-dependent process. Unexpectedly, the complete Snail1 invasion program can be recapitulated by expressing directly either of the membrane-anchored metalloproteinases, MT1-MMP or MT2-MMP. The pro-invasive, angiogenic, and metastatic activities of MT1-MMP and MT2-MMP are unique relative to all other metalloproteinase family members and cannot be mimicked in vivo by the secreted MMPs, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, or MMP-13. Further, siRNA-specific silencing of MT1-MMP and MT2-MMP ablates completely the ability of Snail1 to drive cancer cell BM invasion, induce angiogenesis, or trigger intravasation. Taken together, these data demonstrate that MT1-MMP and MT2-MMP cooperatively function as direct-acting, pro-invasive factors that confer Snaill-triggered cells with the key activities most frequently linked to morbidity and mortality in cancer. EMT | extracellular matrix | Snail www.pnas.org/cgi/doi/10.1073/pnas.0910962106

Autotransporters are a superfamily of virulence factors produced by Gram-negative bacteria consisting of a large N-terminal extra-cellular domain ('passenger domain') and a C-terminal [beta] barrel domain ('[beta] domain'). The mechanism by which the passenger domain is translocated across the outer membrane (OM) is unknown. Here we show that the insertion of a small linker into the passenger domain of the Escherichia coil O157:H7 autotransporter EspP effectively creates a translocation intermediate by transiently stalling translocation near the site of the insertion. Using a site-specific photocrosslinking approach, we found that residues adjacent to the stall point interact with BamA, a component of a heterooligomeric complex (Bam complex) that catalyzes OM protein assembly, and that residues closer to the EspP N terminus interact with the periplasmic chaperones SurA and Skp. The EspP-BamA interaction was short-lived and could be detected only when passenger domain translocation was stalled. These results support a model in which molecular chaperones prevent misfolding of the passenger domain before its secretion and the Bam complex catalyzes both the integration of the[beta] domain into the OM and the translocation of the passenger domain across the OM in a C- to N-terminal direction. autotransporter | Bam complex | outer membrane | protein secretion | virulence factors www.pnas.org/cgi/doi/10.1073/pnas.0907912106

Two distinct bone marrow-derived blast colony-forming cells can generate colonies of lineage-restricted progenitor cells in agar cultures of murine bone marrow. Both cell types selectively had a [Kit.sup.+] [Scal.sup.+] phenotype distinguishing them from most lineage-restricted progenitor ceils. Multicentric blast colony-forming cells stimulated by stem cell factor plus interleukin-6 (IL-6) (BL-CFC-S) were separable from most dispersed blast colony-forming cells stimulated by Flt3 ligand and IL-6 (BL-CFC-F) using CD34 and Flt3R probes. Multicentric BL-CFC-S cofractionated with colony-forming units, spleen (CFU-S) supporting the possibility that the 2 cells may be identical. The colony populations generated by BL-CFC-S were similar in their phenotype and proliferative capacity to progenitor cells in whole bone marrow but the progeny of BL-CFC-F were skewed with an abnormally high proportion of [Kit.sup.-] Flt3[R.sup.+] cells whose clonogenic cells tended to generate only macrophage progeny. Both blast colony populations had a high percentage of GR1+ and Mac[1.sup.+] cells but BL-CFC-F colonies also contained a significant population of B[220.sup.+] and IL-7[R.sup.+] cells relevant to the superior ability of BL-CFC-F colony cells to generate B lymphocytes and the known dependency of this process on Flt3 ligand and IL-7. The commitment events and phenotypic changes during the generation of differing progenitor cells in blast colonies can now be clonally analyzed in a convenient in vitro culture system. blast colonies | lineage commitment | hematopoietic stem cells www.pnas.org/cgi/doi/10.1073/pnas.0910354106

Rab GTPases and their effectors mediate docking, the initial contact of intracellular membranes preceding bilayer fusion. However, it has been unclear whether Rab proteins and effectors are sufficient for intermembrane interactions. We have recently reported reconstituted membrane fusion that requires yeast vacuolar SNAREs, lipids, and the homotypic fusion and vacuole protein sorting (HOPS)/class C Vps complex, an effector and guanine nucleotide exchange factor for the yeast vacuolar Rab GTPase Ypt7p. We now report reconstitution of lysis-free membrane fusion that requires purified GTP-bound Ypt7p, HOPS complex, vacuolar SNAREs, ATP hydrolysis, and the SNARE disassembly catalysts Sec17p and Sec18p. We use this reconstituted system to show that SNAREs and Sec17p/Sec18p, and Ypt7p and the HOPS complex, are required for stable intermembrane interactions and that the three vacuolar Q-SNAREs are sufficient for these interactions. biochemical reconstitution | Rab effector doi/ 10.1073/pnas.0903801106

In several pathological and experimental conditions, the passive membrane conductance of muscle fibers ([G.sub.m]) and their excitability are inversely related. Despite this capacity of [G.sub.m] to determine muscle excitability, its regulation in active muscle fibers is largely unexplored. In this issue, our previous study (Pedersen et al. 2009. J. Gen. Physiol. doi:10.1085/jgp.200910291) established a technique with which biphasic regulation of [G.sub.m] in action potential (AP)-firing fast-twitch fibers of rat extensor digitorum longus muscles was identified and characterized with temporal resolution of seconds. This showed that AP firing initially reduced [G.sub.m] via C1C-1 channel inhibition but after ~1,800 APs, [G.sub.m] rose substantially, causing AP excitation failure. This late increase of [G.sub.m] reflected activation of C1C-1 and KATe channels. The present study has explored regulation of [G.sub.m] in AP-firing slow-twitch fibers of soleus muscle and compared it to [G.sub.m] dynamics in fast-twitch fibers. It further explored aspects of the cellular signaling that conveyed regulation of [G.sub.m] in AP-firing fibers. Thus, in both fiber types, AP firing first triggered protein kinase C (PKC)-dependent C1C-1 channel inhibition that reduced [G.sub.m] by ~50%. Experiments with dantrolene showed that AP-triggered SR [Ca.sup.2+] release activated this PKC-mediated C1C-1 channel inhibition that was associated with reduced rheobase current and improved function of depolarized muscles, indicating that the reduced [G.sub.m] enhanced muscle fiber excitability. In fast-twitch fibers, the late rise in [G.sub.m] was accelerated by glucose-free conditions, whereas it was postponed when intermittent resting periods were introduced during AP firing. Remarkably, elevation of [G.sub.m] was never encountered in AP-firing slow-twitch fibers, even after 15,000 APs. These observations implicate metabolic depression in the elevation of [G.sub.m] in AP-firing fast-twitch fibers. It is concluded that regulation of [G.sub.m] is a general phenomenon in AP-firing muscle, and that differences in [G.sub.m] regulation may contribute to the different phenotypes of fast- and slow-twitch muscle.

Purinergic receptors are expressed in the membrane of the follicular cell layer that communicates with the Xenopus oocyte. Adenosine (Ado) generates a cAMP-dependent [K.sup.+] current ([I.sub.K,cAMP]), whereas ATP activates a [CI.sup.-] current ([F.sub.CI]) and has a dual effect on [I.sub.K.cAMP], provoking both its activation and inhibition. Here, purinergic responses were studied electrophysiologically, first in the whole follicle (w.f.), and then in the same follicle after removal of its epithelium/theca layers (e.t.r. follicle). Responses were analyzed as the ratio of the current amplitudes ([i.sub.etr]/[i.sub.wf]) in the two preparations. For ATP activation of [I.sub.K,cAMP] and [F.sub.CI], the ratios [i.sub.etr]/[i.sub.wf] were 0.053 and 22, respectively, whereas that for Ado was 0.75. Thus, epithelium/theca removal drastically altered the ATP response, suggesting a change in the signaling pathway that correlated with changes in the pharmacological characteristics: the half-maximal effective concentration for activation of the main current in w.f. ([I.sub.K,cAMP]) was 14 [+ or -] 3.8 [micro]M [Hill coefficient (nH) = 2.7 [+ or -] 0.61], and that in e.t.r, follicles ([F.sub.CI]) was 1.8 [+ or -] 0.68 [micro]M (nH = 0.76 [+ or -] 0.09), whereas Ado-response parameters did not change. Responses to UTP and [beta], [gamma]-methylene-ATP, specific agonists for [I.sub.K,cAMP] inhibition and activation, respectively, indicated that in e.t.r, follicles inhibition increased and activation decreased drastically. Thus, purinergic responses were not independent; instead, they were functionally linked. We hypothesize that this property was due to direct interactions between receptors for Ado (A2 subtype) and ATP (P2Y subtype) in the Xenopus follicle. Xenopus oocyte | P2Y receptor | A2 receptor | G protein-coupled receptor oligomerization

Hydrogen sulfide ([H.sub.2]S) has emerged as a new and important member in the group of gaseous signaling molecules. However, the molecular transport mechanism has not yet been identified. Because of structural similarities with [H.sub.2]O, it was hypothesized that aquaporins may facilitate [H.sub.2]S transport across cell membranes. We tested this hypothesis by reconstituting the archeal aquaporin AfAQP from sulfide reducing bacteria Archaeoglobus fulgidus into planar membranes and by monitoring the resulting facilitation of osmotic water flow and [H.sub.2]S flux. To measure [H.sub.2]O and [H.sub.2]S fluxes, respectively, sodium ion dilution and buffer acidification by proton release ([H.sub.2]S [??] [H.sup.+] + H[S.sup.-]) were recorded in the immediate membrane vicinity. Both sodium ion concentration and pH were measured by scanning ion-selective microelectrodes. A lower limit of lipid bilayer permeability to [H.sub.2]S, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] was calculated by numerically solving the complete system of differential reaction diffusion equations and fitting the theoretical pH distribution to experimental pH profiles. Even though reconstitution of AfAQP significantly increased water permeability through planar lipid bilayers, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] remained unchanged. These results indicate that lipid membranes may well act as a barrier to water transport although they do not oppose a significant resistance to [H.sub.2]S diffusion. The fact that cholesterol and sphingomyelin reconstitution did not turn these membranes into an [H.sub.2]S barrier indicates that [H.sub.2]S transport through epithelial barriers, endothelial barriers, and membrane rafts also occurs by simple diffusion and does not require facilitation by membrane channels. aquaporins | gas transport | membrane permeability | unstirred layer | signaling