Search

RNA viruses modify intracellular membranes to produce replication scaffolds. In pancreatic cells, coxsackievirus B3 (CVB3) hijacks membranes from the autophagy pathway, and in vivo disruption of acinar cell autophagy dramatically delays CVB3 replication. This is reversed by expression of GFP-LC3, indicating that CVB3 may acquire membranes from an alternative, autophagy-independent, source(s). Herein, using 3 recombinant CVB3s (rCVB3s) encoding different proteins (proLC3, proLC3(G120A), or ATG4B(C74A)), we show that CVB3 is, indeed, flexible in its utilization of cellular membranes. When compared with a control rCVB3, all 3 viruses replicated to high titers in vivo, and caused severe pancreatitis. Most importantly, each virus appeared to subvert membranes in a unique manner. The proLC3 virus produced a large quantity of LC3-I which binds to phosphatidylethanolamine (PE), affording access to the autophagy pathway. The proLC3(G120A) protein cannot attach to PE, and instead binds to the ER-resident protein SEL1L, potentially providing an autophagy-independent source of membranes. Finally, the ATG4B(C74A) protein sequestered host cell LC3-I, causing accumulation of immature phagophores, and massive membrane rearrangement. Taken together, our data indicate that some RNA viruses can exploit a variety of different intracellular membranes, potentially maximizing their replication in each of the diverse cell types that they infect in vivo.

Summary Ebolavirus NP oligomerizes into helical filaments found at the core of the virion, encapsidates the viral RNA genome, and serves as a scaffold for additional viral proteins within the viral nucleocapsid. We identified a portion of the phosphoprotein homolog VP35 that binds with high affinity to nascent NP and regulates NP assembly and viral genome binding. Removal of the VP35 peptide leads to NP self-assembly via its N-terminal oligomerization arm. NP oligomerization likely causes a conformational change between the NP N- and C-terminal domains, facilitating RNA binding. These functional data are complemented by crystal structures of the NP°-VP35 complex at 2.4 A resolution. The interactions between NP and VP35 illuminated by these structures are conserved among filoviruses and provide key targets for therapeutic intervention.

Although the mechanisms of action (MoA) of nonstructural protein 3 inhibitors (NS3i) and NS5B inhibitors (NS5Bi) are well understood, the MoA of cyclophilin inhibitors (CypI) and NS5A inhibitors (NS5Ai) are not fully defined. In this study, we examined whether CypI and NS5Ai interfere with hepatitis C virus (HCV) RNA synthesis of replication complexes (RCs) or with an earlier step of HCV RNA replication, the creation of double-membrane vesicles (DMVs) essential for HCV RNA replication. In contrast to NS5Bi, both CypI and NS5Ai do not block HCV RNA synthesis by way of RCs, suggesting that they exert their antiviral activity prior to the establishment of enzymatically active RCs. We found that viral replication is not a precondition for DMV formation, since the NS3-NS5B polyprotein or NS5A suffices to create DMVs. Importantly, only CypI and NS5Ai, but not NS5Bi, mir-122, or phosphatidylinositol-4 kinase IIIα (PI4KIIIα) inhibitors, prevent NS3-NS5B-mediated DMV formation. NS3-NS5B was unable to create DMVs in cyclophilin A (CypA) knockdown (KD) cells. We also found that the isomerase activity of CypA is absolutely required for DMV formation. This not only suggests that NS5A and CypA act in concert to build membranous viral factories but that CypI and NS5Ai mediate their early anti-HCV effects by preventing the formation of organelles, where HCV replication is normally initiated. This is the first investigation to examine the effect of a large panel of anti-HCV agents on DMV formation, and the results reveal that CypI and NS5Ai act at the same membranous web biogenesis step of HCV RNA replication, thus indicating a new therapeutic target of chronic hepatitis C.

SummaryProteins, particularly viral proteins, can be multifunctional, but the mechanisms behind multifunctionality are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry, and cellular microscopy that VP40 rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer traffics to the cellular membrane. Once there, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multilayered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle and demonstrate how a single wild-type, unmodified polypeptide can assemble into different structures for different functions.PaperFlick

Macrophages, key cells of the innate immune system, are known to support angiogenesis but are not believed to directly form vessel walls. Here we show that macrophages structurally form primitive, NON-ENDOTHELIAL “vessels” or vascular mimicry (VM) channels in both tumor and angiogenesis in vivo models. These channels are functionally connected to the systemic vasculature as they are perfused by intravenously injected dye. Since both models share hypoxic micro-environments, we hypothesized that hypoxia may be an important mediator of VM formation. Indeed, conditional genetic depletion of myeloid-specific HIF-1α results in decreased VM network formation, dye perfusion and tumor size. Although the macrophage VM network shares some features with an endothelial vasculature, it is ultrastructurally different. Cancer stem cells have been shown to form vascular mimicry channels. Our data demonstrates that tumor-associated macrophages also form them. The identification of this novel type of vascular mimicry may help in the development of targeted cancer therapeutics.

Defining the mechanisms underlying the control of mitochondrial fusion and fission is critical to understanding cellular adaptation to diverse physiological conditions. Here we demonstrate that hypoxia induces fission of mitochondrial membranes, dependent on availability of the mitochondrial scaffolding protein AKAP121. AKAP121 controls mitochondria dynamics through PKA-dependent inhibitory phosphorylation of Drp1 and PKA-independent inhibition of Drp1-Fis1 interaction. Reduced availability of AKAP121 by the ubiquitin ligase Siah2 relieves Drp1 inhibition by PKA and increases its interaction with Fis1, resulting in mitochondrial fission. High AKAP121 levels, seen in cells lacking Siah2, attenuate fission and reduce apoptosis of cardiomyocytes under simulated ischemia. Infarct size and degree of cell death were reduced in Siah2(-/-) mice subjected to myocardial infarction. Inhibition of Siah2 or Drp1 in hatching C. elegans reduces their life span. Through modulating Fis1/Drp1 complex availability, our studies identify Siah2 as a key regulator of hypoxia-induced mitochondrial fission and its physiological significance in ischemic injury and nematode life span.

Autophagy can play an important part in protecting host cells during virus infection, and several viruses have developed strategies by which to evade or even exploit this homeostatic pathway. Tissue culture studies have shown that poliovirus, an enterovirus, modulates autophagy. Herein, we report on in vivo studies that evaluate the effects on autophagy of coxsackievirus B3 (CVB3). We show that in pancreatic acinar cells, CVB3 induces the formation of abundant small autophagy-like vesicles and permits amphisome formation. However, the virus markedly, albeit incompletely, limits the fusion of autophagosomes (and/or amphisomes) with lysosomes, and, perhaps as a result, very large autophagy-related structures are formed within infected cells; we term these structures megaphagosomes. Ultrastructural analyses confirmed that double-membraned autophagy-like vesicles were present in infected pancreatic tissue and that the megaphagosomes were related to the autophagy pathway; they also revealed a highly organized lattice, the individual components of which are of a size consistent with CVB RNA polymerase; we suggest that this may represent a coxsackievirus replication complex. Thus, these in vivo studies demonstrate that CVB3 infection dramatically modifies autophagy in infected pancreatic acinar cells.

Regulated generation of reactive oxygen species (ROS) is primarily accomplished by NADPH oxidases (Nox). Nox1 to Nox4 form a membrane-associated heterodimer with p22(phox), creating the docking site for assembly of the activated oxidase. Signaling specificity is achieved by interaction with a complex network of cytosolic components. Nox4, an oxidase linked to cardiovascular disease, carcinogenesis, and pulmonary fibrosis, deviates from this model by displaying constitutive H(2)O(2) production without requiring known regulators. Extensive Nox4/Nox2 chimera screening was initiated to pinpoint structural motifs essential for ROS generation and Nox subcellular localization. In summary, a matching B loop was crucial for catalytic activity of both Nox enzymes. Substitution of the carboxyl terminus was sufficient for converting Nox4 into a phorbol myristate acetate (PMA)-inducible phenotype, while Nox2-based chimeras never gained constitutive activity. Changing the Nox2 but not the Nox4 amino terminus abolished ROS generation. The unique heterodimerization of a functional Nox4/p22(phox) Y121H complex was dependent on the D loop. Nox4, Nox2, and functional Nox chimeras translocated to the plasma membrane. Cell surface localization of Nox4 or PMA-inducible Nox4 did not correlate with O(2)(-) generation. In contrast, Nox4 released H(2)O(2) and promoted cell migration. Our work provides insights into Nox structure, regulation, and ROS output that will aid inhibitor design.

Talin 1 and 2 connect integrins to the actin cytoskeleton and regulate the affinity of integrins for ligands. In skeletal muscle, talin 1 regulates the stability of myotendinous junctions (MTJs), but the function of talin 2 in skeletal muscle is not known. Here we show that MTJ integrity is affected in talin 2-deficient mice. Concomitant ablation of talin 1 and 2 leads to defects in myoblast fusion and sarcomere assembly, resembling defects in muscle lacking β1 integrins. Talin 1/2-deficient myoblasts express functionally active β1 integrins, suggesting that defects in muscle development are not primarily caused by defects in ligand binding, but rather by disruptions of the interaction of integrins with the cytoskeleton. Consistent with this finding, assembly of integrin adhesion complexes is perturbed in the remaining muscle fibers of talin 1/2-deficient mice. We conclude that talin 1 and 2 are crucial for skeletal muscle development, where they regulate myoblast fusion,sarcomere assembly and the maintenance of MTJs.

Biophysical studies on amyloidogenic and aggregation-prone peptides often require large quantities of material. However, solid-phase synthesis, handling, and purification of peptides often present challenges on these scales. Recombinant expression is an attractive alternative because of its low cost, the ability to isotopically label the peptides, and access to sequences exceeding ~50 residues. However, expression systems that seek to solubilize amyloidogenic peptides suffer from low yields, difficult optimizations, and isolation challenges. We present a general strategy for expressing and isolating amyloidogenic peptides in Escherichia coli by fusion to a polypeptide that drives the expression of attached peptides into bacterial inclusion bodies. This scheme minimizes toxicity during bacterial growth and enables the processing and handling of the peptides in denaturing solutions. Immobilized metal affinity chromatography, reverse phase HPLC, and cyanogen bromide cleavage are used to isolate the peptide, followed by further reverse phase HPLC, which yields milligram quantities of the purified peptide. We demonstrate that driving the peptides into inclusion bodies using fusion to BCL-XL-1/2 is a general strategy for their expression and isolation, as exemplified by the production of 11 peptides species.

Neutrophils kill bacteria on extracellular complexes of DNA fibers and bactericidal proteins known as neutrophil extracellular traps (NETs). The NET composition and the bactericidal mechanisms they use are not fully understood. Here, we show that treatment with deoxyribonuclease (DNase I) impairs a late oxidative response elicited by Gram-positive and Gram-negative bacteria and also by phorbol ester. Isoluminol-dependent chemiluminescence elicited by opsonized Listeria monocytogenes-stimulated neutrophils was inhibited by DNase I, and the DNase inhibitory effect was also evident when phagocytosis was blocked, suggesting that DNase inhibits an extracellular mechanism of reactive oxygen species (ROS) generation. The DNase inhibitory effect was independent of actin polymerization. Phagocytosis and cell viability were not impaired by DNase I. Immunofluorescence analysis shows that myeloperoxidase is present on NETs. Furthermore, granular proteins were detected in NETs from Rab27a-deficient neutrophils which have deficient exocytosis, suggesting that exocytosis and granular protein distribution on NETs proceed by independent mechanisms. NADPH oxidase subunits were also detected on NETs, and the detection of extracellular trap-associated NADPH oxidase subunits was abolished by treatment with DNase I and dependent on cell stimulation. In vitro analyses demonstrate that MPO and NADPH oxidase activity are not directly inhibited by DNase I, suggesting that its effect on ROS production depends on NET disassembly. Altogether, our data suggest that inhibition of ROS production by microorganism-derived DNase would contribute to their ability to evade killing.

The development and function of skeletal muscle depend on molecules that connect the muscle fiber cytoskeleton to the extracellular matrix (ECM). beta1 integrins are ECM receptors in skeletal muscle, and mutations that affect the alpha7beta1 integrin cause myopathy in humans. In mice, beta1 integrins control myoblast fusion, the assembly of the muscle fiber cytoskeleton, and the maintenance of myotendinous junctions (MTJs). The effector molecules that mediate beta1 integrin functions in muscle are not known. Previous studies have shown that talin 1 controls the force-dependent assembly of integrin adhesion complexes and regulates the affinity of integrins for ligands. Here we show that talin 1 is essential in skeletal muscle for the maintenance of integrin attachment sites at MTJs. Mice with a skeletal muscle-specific ablation of the talin 1 gene suffer from a progressive myopathy. Surprisingly, myoblast fusion and the assembly of integrin-containing adhesion complexes at costameres and MTJs advance normally in the mutants. However, with progressive ageing, the muscle fiber cytoskeleton detaches from MTJs. Mechanical measurements on isolated muscles show defects in the ability of talin 1-deficient muscle to generate force. Collectively, our findings show that talin 1 is essential for providing mechanical stability to integrin-dependent adhesion complexes at MTJs, which is crucial for optimal force generation by skeletal muscle.

Methods for the construction of ordered nanoscale arrays have been implicated in fields ranging from separation technologies to microelectronics. Yet, despite the plethora of nanoscale structures assembled in nature that use a templating strategy, chemists have been unable to replicate this success. A technology is reported for templated organic polymers composed of filamentous bacteriophage-polyacrylamide biomacromolecules that self-assemble into highly ordered helical bundles displaying hexagonal close packing. The results align with a previously reported mathematical prediction for the close packing of flexible tubes. This biopolymeric assembly can be viewed as a magnification of the inherent microscopic chirality and helicity present in individual phage particles at the macroscale level.

Ligand-binding epitopes of proteins can mutate rapidly, as shown by viral mutations that lead to escape from neutralizing antibodies. We have undertaken to recreate in vitro the evolutionary competition between viral mutations that allow escape from antibody binding and host mutations that generate new neutralizing antibodies to the mutated viral antigen. To examine this vital race, we describe a phage-based method that allows rapid analysis of molecules that perturb the binding of proteins to their ligands. Because the system can amplify by replication, single-molecule sensitivity can be achieved. When combinatorial protein or small-molecule libraries are studied, large numbers of binding events can be analyzed simultaneously. Such libraries may be used in a sequential phage escape format, where cycles of phage binding and release of mutants are driven by antibodies or small molecules and the difficulty of escape increases at each cycle. Ultimately, the sequencing of the viral mutants allows annotation of the allowed trajectory of escape. Likewise, sequencing of the antibody perturbants charts the chemistry of the immune system response to the viral challenge. We have termed such analysis of competing mutations a “checkmate analysis.” When viral systems are studied, a checkmate analysis allows experimental evaluation of the evolutionary contest between viruses and the immune system and may predict which antibodies and small-molecule ligands should be generated in anticipation of viral mutations before these mutations create viral epidemics.

Cells of the monocyte/macrophage lineage have been shown to be the principal targets for productive HIV-1 replication within the CNS. In addition, HIV-1-associated dementia (HAD) has been shown to correlate with macrophage abundance in the brain. Although increased entry of monocytes into the brain is thought to initiate this process, mechanisms that prevent macrophage egress from the brain and means that prevent macrophage death may also contribute to cell accumulation. We hypothesized that osteopontin (OPN) was involved in the accumulation of macrophages in the brain in neuroAIDS. Using in vitro model systems, we have demonstrated the role of OPN in two distinct aspects of macrophage accumulation: prevention from recirculation and protection from apoptosis. In these unique mechanisms, OPN would aid in macrophage survival and accumulation in the brain, the pathological substrate of HAD.

Development of a prophylactic vaccine against hepatitis C virus (HCV) has been hampered by the extraordinary viral diversity and the poor host immune response. Scaffolding, by grafting an epitope onto a heterologous protein scaffold, offers a possible solution to epitope vaccine design. In this study, we designed and characterized epitope vaccine antigens for the antigenic sites of HCV envelope glycoproteins E1 (residues 314–324) and E2 (residues 412–423), for which neutralizing antibody-bound structures are available. We first combined six structural alignment algorithms in a “scaffolding meta-server” to search for diverse scaffolds that can structurally accommodate the HCV epitopes. For each antigenic site, ten scaffolds were selected for computational design and the resulting epitope scaffolds were analyzed using structure-scoring functions and molecular dynamics simulation. We experimentally confirmed that three E1 and five E2 epitope scaffolds bound to their respective neutralizing antibodies, but with different kinetics. We then investigated a “multivalent scaffolding” approach by displaying 24 copies of an epitope scaffold on a self-assembling nanoparticle, which markedly increased the avidity of antibody binding. Our study thus demonstrates the utility of a multi-scale scaffolding strategy in epitope vaccine design and provides promising HCV immunogens for further assessment in vivo.

Fluorocarbons are lipophobic and non-polar molecules that exhibit remarkable bio-compatibility, with applications in liquid ventilation and synthetic blood. The unique properties of these compounds have also enabled mass spectrometry imaging of tissues where the fluorocarbons act as a Teflon-like coating for nanostructured surfaces to assist in desorption/ionization. Here we report fluorinated gold nanoparticles (f-AuNPs) designed to facilitate nanostructure imaging mass spectrometry. Irradiation of f-AuNPs results in the release of the fluorocarbon ligands providing a driving force for analyte desorption. The f-AuNPs allow for the mass spectrometry analysis of both lipophilic and polar (central carbon) metabolites. An important property of AuNPs is that they also act as contrast agents for X-ray microtomography and electron microscopy, a feature we have exploited by infusing f-AuNPs into tissue via fluorocarbon liquids to facilitate multi-modal (molecular and anatomical) imaging.

Summary The emergence of antibiotic resistance places a sense of urgency on the development of alternative antibacterial strategies, of which targeting virulence factors has been regarded as a "second generation" antibiotic approach. In the case of Pseudomonas aeruginosa infections, a proteolytic virulence factor, LasB, is one such target. Unfortunately, we and others have not been successful in translating in vitro potency of LasB inhibitors to in vivo efficacy in an animal model. To overcome this obstacle, we now integrate in silico and in vitro identification of the mercaptoacetamide motif as an effective class of LasB inhibitors with full in vivo characterization of mercaptoacetamide prodrugs using Caenorhabditis elegans . We show that one of our mercaptoacetamide prodrugs has a good selectivity profile and high in vivo efficacy, and confirm that LasB is a promising target for the treatment of bacterial infections. In addition, our work highlights that the C. elegans infection model is a user-friendly and cost-effective translational tool for the development of anti-virulence compounds.

In the developing myocardium, vascular endothelial growth factor (VEGF)–dependent neovascularization occurs by division of existing vessels, a process that persists for several weeks following birth. During this remodeling phase, mRNA expression of β3 integrin in the heart decreases significantly as vessel maturation progresses. However, in male mice lacking β3, coronary capillaries fail to mature and continue to exhibit irregular endothelial thickness, endothelial protrusions into the lumen, and expanded cytoplasmic vacuoles. Surprisingly, this phenotype was not seen in female β3-null mice. Enhanced VEGF signaling contributes to the β3-null phenotype, because these vessels can be normalized by inhibitors of VEGF or Flk-1. Moreover, intravenous injection of VEGF induces a similar angiogenic phenotype in hearts of adult wild-type mice. These findings show a clear vascular phenotype in the hearts of mice lacking β3 and suggest this integrin plays a critical role in coronary vascular development and the vascular response to VEGF.

The water channel protein PvTIP3;1 (α-TIP) is a member of the m ajor i ntrinsic p rotein (MIP) membrane channel family. We overexpressed this eukaryotic aquaporin in the methylotrophic yeast Pichia pastoris , and immunogold labeling of cellular cryosections showed that the protein accumulated in the plasma membrane, as well as vacuolar and other intracellular membranes. We then developed an in vivo functional assay for water channel activity that measures the change in optical absorbance of spheroplasts following an osmotic shock. Spheroplasts of wild-type P. pastoris displayed a linear relationship between absorbance and osmotic shock level. However, spheroplasts of P. pastoris expressing PvTIP3;1 showed a break in this linear relationship corresponding to hypo-osmotically induced lysis. It is the difference between control and transformed spheroplasts under conditions of hypo-osmotic shock that forms the basis of our aquaporin activity assay. The aquaporin inhibitor mercury chloride blocked water channel activity but had no effect on wild-type yeast. Osmotically shocked yeast cells were affected only slightly by expression of the Escherichia coli glycerol channel GlpF, which belongs to the MIP family but is a weak water channel. The important role that aquaporins play in human physiology has led to a growing interest in their potential as drug targets for treatment of hypertension and congestive heart failure, as well as other fluid overload states. The simplicity of this assay that is specific for water channel activity should enable rapid screening for compounds that modulate water channel activity.

Oxidative stress is implicated in a number of neurological disorders including stroke, Parkinson's disease, and Alzheimer's disease. To study the effects of oxidative stress on neuronal cells, we have used an immortalized mouse hippocampal cell line (HT-22) that is particularly sensitive to glutamate. In these cells, glutamate competes for cystine uptake, leading to a reduction in glutathione and, ultimately, cell death. As it has been reported that protein kinase C activation inhibits glutamate toxicity in these cells and is also associated with the inhibition of apoptosis in other cell types, we asked if glutamate toxicity was via apoptosis. Morphologically, glutamate-treated cells underwent plasma membrane blebbing and cell shrinkage, but no DNA fragmentation was observed. At the ultrastructural level, there was damage to mitochondria and other organelles although the nuclei remained intact. Protein and RNA synthesis inhibitors as well as certain protease inhibitors protected the cells from glutamate toxicity. Both the macromolecular synthesis inhibitors and the protease inhibitors had to be added relatively soon after the addition of glutamate, suggesting that protein synthesis and protease activation are early and distinct steps in the cell death pathway. Thus, the oxidative stress brought about by treatment with glutamate initiates a series of events that lead to a form of cell death distinct from either necrosis or apoptosis.

Complex hereditary spastic paraplegia (HSP) is a genetic disorder that causes lower limb spasticity and weakness and intellectual disability. Deleterious mutations in the poorly characterized serine hydrolase DDHD2 are a causative basis for recessive complex HSP. DDHD2 exhibits phospholipase activity in vitro, but its endogenous substrates and biochemical functions remain unknown. Here, we report the development of DDHD2(-/-) mice and a selective, in vivo-active DDHD2 inhibitor and their use in combination with mass spectrometry-based lipidomics to discover that DDHD2 regulates brain triglycerides (triacylglycerols, or TAGs). DDHD2(-/-) mice show age-dependent TAG elevations in the central nervous system, but not in several peripheral tissues. Large lipid droplets accumulated in DDHD2(-/-) brains and were localized primarily to the intracellular compartments of neurons. These metabolic changes were accompanied by impairments in motor and cognitive function. Recombinant DDHD2 displays TAG hydrolase activity, and TAGs accumulated in the brains of wild-type mice treated subchronically with a selective DDHD2 inhibitor. These findings, taken together, indicate that the central nervous system possesses a specialized pathway for metabolizing TAGs, disruption of which leads to massive lipid accumulation in neurons and complex HSP syndrome.

When outfitted with long alkyl chains, polycaps, capsules along a polymer chain, spontaneously organize themselves into a two-dimensional liquid crystalline phase. Further organization results from shearing or pulling the liquid crystalline samples, producing three-dimensional assemblies of micrometer-wide, infinitely long fibers (see schematic representation).

The interaction of the non-receptor tyrosine kinase, Src, with the cytoskeleton of adhesion sites was studied in nerve growth cones isolated from fetal rat brain. Of particular interest was the role of protein tyrosine phosphatases in the regulation of Src-cytoskeleton binding. Growth cones were found to contain a high level of protein tryrosine phosphatase activity, most of it membrane-associated and forming large, multimeric and wheat germ agglutinin-binding complexes. The receptor tyrosine phosphatase PTPalpha seems to be the most prevalent species among the membrane-associated enzymes. As seen by immunofluorescence, PTPalpha is present throughout the plasmalemma of the growth cone including filopodia, and it forms a punctate pattern consistent with that of integrin beta1. For adhesion site analysis, isolated growth cones were either plated onto the neurite growth substratum, laminin, or kept in suspension. Plating growth cones on laminin triggered an 8-fold increase in Src binding to the adherent cytoskeleton. This effect was blocked completely with the protein tyrosine phosphatase inhibitor, vanadate. Growth cone plating also increased the association with adhesion sites of tyrosine phosphatase activity (14-fold) and of PTPalpha immunoreactivity (6-fold). Vanadate blocked the enzyme activity but not the recruitment of PTPalpha to the adhesion sites. In conjunction with our previous results on growth cones, these data suggest that integrin binding to laminin triggers the recruitment of PTPalpha (and perhaps other protein tyrosine phosphatases) to adhesion sites, resulting in de-phosphorylation of Src's tyr 527. As a result Src unfolds, becomes kinase-active, and its SH2 domain can bind to an adhesion site protein. This implies a critical role for protein tyrosine phosphatase activity in the earliest phases of adhesion site assembly.

Summary The movement protein (MP) of tobacco mosaic virus (TMV) facilitates the cell-to-cell spread of infection by altering the structure and function of plasmodesmata, the intercellular communication channels in plants. Because the protein was shown to interfere with intercellular communication when expressed in the cyanobacterium Anabaena sp. strain PCC 7120, whether the ability of the protein to target and to modify intercellular communication channels in plants is conserved in this prokaryote was investigated. It was found that the MP localizes to the cell junctions and induces the formation of filamentous structures that traverse the septa. It is proposed that the protein interacts with host components that are similar between plants and Anabaena and that may be evolutionarily related. The observations in Anabaena suggest that the MP modifies plasmodesmata by forming a filamentous aggregate within the pore.

Methamphetamine (Meth) abuse has been shown to induce alterations in mitochondrial function in the brain as well as to induce hyperthermia, which contributes to neurotoxicity and Meth-associated mortality. Brown adipose tissue (BAT), a thermogenic site known to be important in neonates, has recently regained importance since being identified in significant amounts and in correlation with metabolic balance in human adults. Given the high mitochondrial content of BAT and its role in thermogenesis, we aimed to investigate whether BAT plays any role in the development of Meth-induced hyperthermia. By ablating or denervating BAT, we identified a partial contribution of this organ to Meth-induced hyperthermia. BAT ablation decreased temperature by 0.5°C and reduced the length of hyperthermia by 1 h, compared to sham-operated controls. BAT denervation also affected the development of hyperthermia in correlation with decreased the expression of electron transport chain molecules, and increase on PCG1a levels, but without affecting Meth-induced uncoupling protein 1 upregulation. Furthermore, in isolated BAT cells in culture, Meth, but not Norepinephrine, induced H2O2 upregulation. In addition, we found that in vivo Reactive Oxygen Species (ROS) play a role in Meth hyperthermia. Thus, sympathetically mediated mitochondrial activation in the BAT and Meth-induced ROS are key components to the development of hyperthermia in Meth abuse.

The investigation of the molecular properties of nerve growth cones depends to a significant degree on their isolation from fetal brain in the form of 'growth cone particles' (GCPs). The availability of markers for developing axons and dendrites, as well as glial cells, has made it possible to characterize the GCP fraction in much greater detail than before and to optimize its yield. Marker analyses show that a member of the N-CAM family (5B4-CAM), synaptophysin, and especially GAP-43 and non-phosphorylated tau, are enriched in the GCP fraction. In contrast, MAP2 and, particularly, glial fibrillary acidic protein and vimentin are fractionated away from GCPs. Furthermore, GCP yield can be doubled relative to the original procedure, without compromising purity, by raising the sucrose concentration of the fractionation gradient's uppermost layer. The results indicate that GCPs are highly purified growth cone fragments with very little glial contamination, and that they are primarily of axonal origin.

Previous saxitoxin binding studies indicated two forms of the sodium channel in the fetal rat brain; a low-affinity precursor located in an internal membrane compartment, present exclusively in growth cones and a high-affinity mature form present in the plasmalemma of growth cones and characteristic of synapses. This raises the questions (1) of the presence or absence of the beta2 subunit in these channel forms and (2) of the developmental regulation of the the beta2 subunit. Antibodies against the alpha and beta2 channel subunits were used to probe Western blots of subcellular fractions from rat brains at embryonic day 18 (E18), pups at postnatal (P) days 7-25, and adults, as well as purified sodium channels from adult brain. In both synaptosomes and the purified sodium channel the beta2 antibody recognized the expected band at 38 kDa under reducing conditions. However, in contrast to the alpha subunit, this band was absent at E18 and became apparent only from P7 onwards. At the earlier time intervals a very prominent immunoreactive band of unknown identity was evident at 260--300kDa, which declined in intensity concomitant with the appearance of the 38 kDa beta2 band. These data indicate that beta2 subunits are regulated independently from alpha subunits, are absent in differentiating neurons, and hence are not necessary for insertion of the sodium channel into the plasmalemma, at least during early development of the neuron.

The mechanisms of action by which cyclophilin inhibitors (CypI) interfere with the HCV life cycle remain poorly understood. We reported that CypI and NS5A inhibitors (NS5Ai), but not other classes of anti-HCV agents, prevent assembly of double membrane vesicles (DMVs), which protect replication complexes. We demonstrated that both NS5A and the isomerase cyclophilin A (CypA) are required for DMV formation. Here, we examined whether CypI mediate an additional antiviral effect that could further explain the high efficacy of CypI. We identified a unique action of CypI. CypI remodel the organization of the endoplasmic reticulum (ER) of HCV-infected cells, but not of uninfected cells. This effect is specific since it was not observed for other classes of anti-HCV agents including NS5Ai, and has no effect on the viability of CypI-treated cells. Since ER serves as platform for the establishment of HCV replication complexes, we asked whether the ER reorganization by CypI would prevent cells from being newly infected. Remarkably, CypI-treated HCV-pre-infected cells remain totally impervious to a reinfection, suggesting that the CypI-mediated ER reorganization prevents a reinfection. This block is not due to residual CypI since CypI-resistant HCV variants also fail to infect these cells. The ER reorganization by CypI is rapid and reversible. This study provides the first evidence that CypI trigger a unique ER reorganization of infected cells, rendering cells transiently impervious to a reinfection. This study further suggests that the HCV-induced ER rearrangement represents a key target for the development of new therapies.

Acute myocardial infarction (MI), which involves the rupture of existing atheromatous plaque, remains highly unpredictable despite recent advances in the diagnosis and treatment of coronary artery disease. Accordingly, a clinical measurement that can predict an impending MI is desperately needed. Here, we characterize circulating endothelial cells (CECs) using an automated and clinically feasible CEC three-channel fluorescence microscopy assay in 50 consecutive patients with ST-segment elevation MI and 44 consecutive healthy controls. CEC counts were significantly elevated in MI cases versus controls, with median numbers of 19 and 4 cells/ml, respectively (P = 1.1 × 10(-10)). A receiver-operating characteristic (ROC) curve analysis demonstrated an area under the ROC curve of 0.95, suggesting near-dichotomization of MI cases versus controls. We observed no correlation between CECs and typical markers of myocardial necrosis (ρ = 0.02, creatine kinase-myocardial band; ρ = -0.03, troponin). Morphological analysis of the microscopy images of CECs revealed a 2.5-fold increase (P < 0.0001) in cellular area and a twofold increase (P < 0.0001) in nuclear area of MI CECs versus healthy controls, age-matched CECs, as well as CECs obtained from patients with preexisting peripheral vascular disease. The distribution of CEC images that contained from 2 to 10 nuclei demonstrates that MI patients were the only subject group to contain more than 3 nuclei per image, indicating that multicellular and multinuclear clusters are specific for acute MI. These data indicate that CEC counts may serve as a promising clinical measure for the prediction of atherosclerotic plaque rupture events.

Deficiencies of subunits of the transcriptional regulatory complex Mediator generally result in embryonic lethality, precluding study of its physiological function. Here we describe a missense mutation in Med30 causing progressive cardiomyopathy in homozygous mice that, although viable during lactation, show precipitous lethality 2–3 wk after weaning. Expression profiling reveals pleiotropic changes in transcription of cardiac genes required for oxidative phosphorylation and mitochondrial integrity. Weaning mice to a ketogenic diet extends viability to 8.5 wk. Thus, we establish a mechanistic connection between Mediator and induction of a metabolic program for oxidative phosphorylation and fatty acid oxidation, in which lethal cardiomyopathy is mitigated by dietary intervention.

Disruption of autophagy--a key homeostatic process in which cytosolic components are degraded and recycled through lysosomes--can cause neurodegeneration in tissue culture and in vivo. Upregulation of this pathway may be neuroprotective, and much effort is being invested in developing drugs that cross the blood brain barrier and increase neuronal autophagy. One well-recognized way of inducing autophagy is by food restriction, which upregulates autophagy in many organs including the liver; but current dogma holds that the brain escapes this effect, perhaps because it is a metabolically privileged site. Here, we have re-evaluated this tenet using a novel approach that allows us to detect, enumerate and characterize autophagosomes in vivo. We first validate the approach by showing that it allows the identification and characterization of autophagosomes in the livers of food-restricted mice. We use the method to identify constitutive autophagosomes in cortical neurons and Purkinje cells, and we show that short-term fasting leads to a dramatic upregulation in neuronal autophagy. The increased neuronal autophagy is revealed by changes in autophagosome abundance and characteristics, and by diminished neuronal mTOR activity in vivo, demonstrated by a reduction in levels of phosphorylated S6 ribosomal protein in Purkinje cells. The increased abundance of autophagosomes in Purkinje cells was confirmed using transmission electron microscopy. Our data lead us to speculate that sporadic fasting might represent a simple, safe and inexpensive means to promote this potentially therapeutic neuronal response.

We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

Familial amyloidosis of Finnish type (FAF) is a systemic amyloid disease associated with the deposition of proteolytic fragments of mutant (D187N/Y) plasma gelsolin. We report a mouse model of FAF featuring a muscle-specific promoter to drive D187N gelsolin synthesis. This model recapitulates the aberrant endoproteolytic cascade and the aging-associated extracellular amyloid deposition of FAF. Amyloidogenesis is observed only in tissues synthesizing human D187N gelsolin, despite the presence of full-length D187N gelsolin and its 68-kDa cleavage product in blood—demonstrating the importance of local synthesis in FAF. Loss of muscle strength was progressive in homozygous D187N gelsolin mice. The presence of misfolding-prone D187N gelsolin appears to exacerbate the age-associated decline in cellular protein homeostasis (proteostasis), reflected by the intracellular deposition of numerous proteins, a characteristic of the most common degenerative muscle disease of aging humans, sporadic inclusion body myositis.

Bacillus anthracis lethal toxin (LT) is a key virulence factor of anthrax and contributes significantly to the in vivo pathology. The enzymatically active component is a Zn(2+)-dependent metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MKKs). Using ex vivo differentiated human lung epithelium we report that LT destroys lung epithelial barrier function and wound healing responses by immobilizing the actin and microtubule network. Long-term exposure to the toxin generated a unique cellular phenotype characterized by increased actin filament assembly, microtubule stabilization, and changes in junction complexes and focal adhesions. LT-exposed cells displayed randomly oriented, highly dynamic protrusions, polarization defects and impaired cell migration. Reconstitution of MAPK pathways revealed that this LT-induced phenotype was primarily dependent on the coordinated loss of MKK1 and MKK2 signaling. Thus, MKKs control fundamental aspects of cytoskeletal dynamics and cell motility. Even though LT disabled repair mechanisms, agents such as keratinocyte growth factor or dexamethasone improved epithelial barrier integrity by reducing cell death. These results suggest that co-administration of anti-cytotoxic drugs may be of benefit when treating inhalational anthrax.

Innate immune system receptors function as sensors of infection and trigger the immune responses through ligand-specific signaling pathways. These ligands are pathogen-associated products, such as components of bacterial walls and viral nuclear acids. A common response to such ligands is the activation of mitogen-activated protein kinase p38, whereas double-stranded viral RNA additionally induces the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha). Here we have shown that p38 and eIF2alpha phosphorylation represent two biochemical markers of the effects induced by N-(3-oxo-acyl)homoserine lactones, the secreted products of a number of Gram-negative bacteria, including the human opportunistic pathogen Pseudomonas aeruginosa. Furthermore, N-(3-oxo-dodecanoyl)homoserine lactone induced distension of mitochondria and the endoplasmic reticulum as well as c-jun gene transcription. These effects occurred in a wide variety of cell types including alveolar macrophages and bronchial epithelial cells, requiring the structural integrity of the lactone ring motif and its natural stereochemistry. These findings suggest that N-(3-oxo-acyl)homoserine lactones might be recognized by receptors of the innate immune system. However, we provide evidence that N-(3-oxo-dodecanoyl)homoserine lactone-mediated signaling does not require the presence of the canonical innate immune system receptors, Toll-like receptors, or two members of the NLR/Nod/Caterpillar family, Nod1 and Nod2. These data offer a new understanding of the effects of N-(3-oxo-dodecanoyl)homoserine lactone on host cells and its role in persistent airway infections caused by P. aeruginosa.

Pulmonary pathologies including adult respiratory distress syndrome are characterized by disruption of pulmonary integrity and edema compromising respiratory function. Sphingosine 1-phosphate (S1P) is a lipid mediator synthesized and/or stored in mast cells, platelets, and epithelial cells, with production up-regulated by the proinflammatory cytokines IL-1 and TNF. S1P administration via the airways but not via the vasculature induces lung leakage. Using receptor-null mice, we show that S1P, acting on S1P3receptor expressed on both type I and type II alveolar epithelial cells but not vascular endothelium, induces pulmonary edema by acute tight junction opening. WT but not S1P3-null mice showed disruption of pulmonary epithelial tight junctions and the appearance of paracellular gaps between epithelial cells by electron microscopy within 1 h of airways exposure to S1P. We further show by fluorescence microscopy that S1P induced rapid loss of ZO-1 reactivity, an essential component of the cytoplasmic plaque associated with tight junctions, as well as of the tetraspannin Claudin-18, an integral membrane organizer of tight junctions. S1P shows synergistic activity with the proinflammatory cytokine TNF, showing both pulmonary edema and mortality at subthreshold S1P doses. Specifically, preexposure of mice to subthreshold doses of TNF, which alone induced no lung edema, exacerbated S1P-induced edema and impaired survival. S1P, acting through S1P3, regulates epithelial integrity and acts additively with TNF in compromising respiratory barrier function. Because S1P3-null mice are resistant to S1P-induced pulmonary leakage, either alone or in the presence of TNF, S1P3antagonism may be useful in protecting epithelial integrity in pulmonary disease.

Many heat-shock proteins (Hsp) are members of evolutionarily conserved families of chaperone proteins that inhibit the aggregation of unfolded polypeptides and refold denatured proteins, thereby remedying phenotypic effects that may result from protein aggregation or protein instability. Here we report that the mitochondrial chaperone Hsp40, also known as Dnaja3 or Tid1, is differentially expressed during cardiac development and pathological hypertrophy. Mice deficient in Dnaja3 developed dilated cardiomyopathy (DCM) and died before 10 weeks of age. Progressive respiratory chain deficiency and decreased copy number of mitochondrial DNA were evident in cardiomyocytes lacking Dnaja3. Profiling of Dnaja3-interacting proteins identified the alpha-subunit of DNA polymerase gamma (Polga) as a client protein. These findings suggest that Dnaja3 is crucial for mitochondrial biogenesis, at least in part, through its chaperone activity on Polga and provide genetic evidence of the necessity for mitochondrial Hsp40 in preventing DCM.

A novel cytoplasmic compartment referred to as GW bodies was initially identified using human autoantibodies to a 182 kDa protein named GW182. GW bodies are small, generally spherical, cytoplasmic domains that vary in number and size in several mammalian cell types examined to date. Based on our earlier studies, GW bodies were proposed to be cytoplasmic sites for mRNA storage and/or degradation. In the present study, immunogold electron microscopy identified electron dense structures of 100-300 nm diameter devoid of a lipid bilayer membrane. These structures appeared to comprise clusters of electron dense strands of 8-10 nm in diameter. By costaining with CENP-F and PCNA, and employing a double-thymidine block to synchronize HeLa cells, GW bodies were observed to be small in early S phase and larger during late S and G2 phases of the cell cycle. The majority of GW bodies disassembled prior to mitosis and small GW bodies reassembled in early G1. The analysis of GW bodies in two experimental models of cell proliferation using reversal of 3T3/serum-starvation and concanavalin A stimulation of mouse splenocytes and T cells, revealed that proliferating cells contained larger, brighter, and more numerous GW bodies as well as up to a fivefold more total GW182 protein than quiescent cells. In vitro gene knockdown of GW182 led to the disappearance of GW bodies demonstrating that GW182 is a critical component of GW bodies. The incremental expression of the GW182 protein in cells induced to proliferate and the cyclic formation and breakdown of GW bodies during mitosis are intriguing in view of the notion that GW bodies are specialized centers involved in maintaining stability and/or controlling degradation of mRNA.

Pyrrole-imidazole polyamides bind DNA with affinities comparable to those of transcriptional regulatory proteins and inhibit the DNA binding activities of components of the transcription apparatus. If polyamides are to be useful for the regulation of gene expression in cell culture experiments, one pivotal issue is accessibility of specific sites in nuclear chromatin. We first determined the kinetics of uptake and subcellular distribution of polyamides in lymphoid and myeloid cells using fluorescent polyamide-bodipy conjugates and deconvolution microscopy. Then cells were incubated with a polyamide-chlorambucil conjugate, and the sites of specific DNA cleavage in the nuclear chromatin were assayed by ligation-mediated PCR. In addition, DNA microarray analysis revealed that two different polyamides generated distinct transcription profiles. Remarkably, the polyamides affected only a limited number of genes.

Proteins, particularly viral proteins, can be multifunctional, but the mechanism(s) behind this trait are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry and cellular microscopy that the Ebola virus VP40 protein rearranges into different structures, each with a distinct and essential function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer trafficks to the cellular membrane. There, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multi-layered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly, but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle, and demonstrate how a single, wild-type, unmodified polypeptide can assemble into different structures for different functions.

The amino acid, L-arginine (L-Arg), is a potent taste stimulus for the channel catfish, Ictalurus punctatus. Receptor binding studies demonstrated a high-affinity binding of L-Arg to putative taste receptor sites. This binding could be inhibited by preincubation of the tissue in the lectins Phaseolus vulgaris agglutinin (PHA) and Ricinus communis agglutinin I (RCA I). Neurophysiological studies demonstrated that the L-Arg receptor is a stimulus-gated ion channel type receptor whose conductance was stimulated by L-Arg and inhibited by D-arginine (D-Arg). To purify the receptor we subjected CHAPS solubilized partial membrane preparation from barbel epithelium to RCA I lectin affinity chromatography. The bound proteins were eluted with D-galactose. When these proteins were reconstituted into lipid bilayers, L-Arg activated single channel currents with conductances between 45 and 85 pS. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted protein showed a distinct band at approximately 83 kDa. Polyclonal antibodies raised against this 83-kDa band in guinea pigs reacted with numerous small (approximately 1 micron) sites within the taste pore of every taste bud when applied to fixed nonpermeabilized barbels. This observation suggests that the antibodies recognize an externally-facing epitope of the putative Arg receptor. The antibodies also inhibited L-Arg-stimulated currents in reconstitution studies. Sephacryl S-300 HR chromatography of the eluant from the affinity column showed a high molecular weight peak (> 700 kDa) which was recognized by the antibodies. Reconstitution of the protein from this peak into a lipid bilayer resulted in L-Arg-stimulated channels that could be inhibited by D-Arg. This high molecular weight component may be aggregates of the arginine taste receptor.