Mechanisms of Toxicity of Ag Nanoparticles in Comparison to Bulk and Ionic Ag on Mussel Hemocytes and Gill Cells

Silver nanoparticles (Ag NPs) are increasingly used in many products and are expected to end up in the aquatic environment. Mussels have been proposed as marine model species to evaluate NP toxicity in vitro. The objective of this work was to assess the mechanisms of toxicity of Ag NPs on mussel hemocytes and gill cells, in comparison to ionic and bulk Ag. Firstly, cytotoxicity of commercial and maltose stabilized Ag NPs was screened in parallel with the ionic and bulk forms at a wide range of concentrations in isolated mussel cells using cell viability assays. Toxicity of maltose alone was also tested. LC50 values were calculated and the most toxic Ag NPs tested were selected for a second step where sublethal concentrations of each Ag form were tested using a wide array of mechanistic tests in both cell types. Maltose-stabilized Ag NPs showed size-dependent cytotoxicity, smaller (20 nm) NPs being more toxic than larger (40 and 100 nm) NPs. Maltose alone provoked minor effects on cell viability. Ionic Ag was the most cytotoxic Ag form tested whereas bulk Ag showed similar cytotoxicity to the commercial Ag NPs. Main mechanisms of action of Ag NPs involved oxidative stress and genotoxicity in the two cell types, activation of lysosomal AcP activity, disruption of actin cytoskeleton and stimulation of phagocytosis in hemocytes and increase of MXR transport activity and inhibition of Na-K-ATPase in gill cells. Similar effects were observed after exposure to ionic and bulk Ag in the two cell types, although generally effects were more marked for the ionic form. In conclusion, results suggest that most observed responses were due at least in part to dissolved Ag.

Estudo da sinalização da insulina nos tecidos muscular e adiposo de ratos submetidos à desnutrição materna no início da lactação

Vários estudos sugerem que a desnutrição materna no período pós-natal poderia causar alterações na homeostase glicêmica da prole na vida adulta. Neste trabalho objetivamos investigar a interferência da programação metabólica induzida pela desnutrição protéica materna durante o início da lactação sobre a homeostase glicêmica e a sinalização da insulina nos tecidos muscular e adiposo. Animais desnutridos (D-dieta da mãe contendo 0% de proteína nos primeiros 10 dias de lactação) ou controle (C-dieta da mãe contendo 22% de proteína) foram estudados do nascimento até a vida adulta. Em resumo, observamos uma diminuição na insulina plasmática acompanhada de normoglicemia nos animais adultos desnutridos. A ativação do receptor de insulina (IR), após a estimulação com o hormônio apresentou-se diminuída durante o período de restrição protéica em músculo isolado destes animais experimentais. Durante o período da lactação, observamos uma diminuição na captação de glicose, na fosforilação do substrato para o receptor de insulina (IRS 1) e na translocação do GLUT 4 no tecido muscular. Na idade adulta, entretanto, houve aumento significativo na captação de glicose e translocação do GLUT 4 no músculo, associado com o aumento na expressão da PI3 quinase associada ao IRS 1. No tecido adiposo de ratos desnutridos adultos observamos menor fosforilação em tirosina tanto do IR quanto do IRS 1, que foi compensada pela maior ativação do IRS 2 e da PI3 quinase. Os níveis basais de pAkt e de GLUT 4 na membrana estavam aumentados, culminando em um aumento na captação de glicose. Observamos também uma redistribuição do citoesqueleto de actina e maior resistência aos efeitos da Ltrunculina B nos adipócitos dos ratos desnutridos. Em conclusão, este estudo demonstrou que a desnutrição materna no início da lactação é capaz de causar alterações na prole na vida adulta, o que parece estar relacionado com a expressão e ativação de proteínas chave na cascata da sinalização da insulina nos tecidos periféricos, importantes na regulação do metabolismo da glicose.

Dose terapêutica de radiação ionizante induz um fenótipo migratório em células de carcinoma de colon humano (caco-2): vias de sinalização envolvidas

O câncer colo-retal é a terceira neoplasia mais frequente em todo o mundo e a recorrência local e neoplasia refratária são desafios no tratamento do câncer colo-retal após a cirurgia convencional. Com o intuito de controlar a recorrência e aumentar a média de sobrevida dos pacientes, uma estratégia multidisciplinar que combina a radioterapia (RT) e a quimioterapia com o processo cirúrgico tem sido protocolo clínico de escolha. Embora esta combinação seja capaz de otimizar o tratamento, nem todos os pacientes são beneficiados com o protocolo quimio-rádio combinado, uma vez que existem os insucessos terapêuticos relacionados com a incidência de neoplasias secundárias tardias em pacientes que foram submetidos à RT para tratamento de neoplasias anteriores. Além da doença refratária, outro agravante da RT são os efeitos colaterais produzidos pela radiação ionizante (IR), em especial àqueles do trato gastrointestinal. Estes efeitos estão relacionados com alterações da homeostase do epitélio intestinal, através da desorganização dos complexos juncionais. Porém, os mecanismos que medeiam estes efeitos ainda não estão elucidados. Este estudo avaliou as vias de sinalização que medeiam os efeitos da IR em células Caco-2. Foi observado que a IR causa uma desorganização da junção aderente via Src, EGFR e MAPK, sendo estas alterações acompanhadas por desorganização do citoesqueleto de actina em todo o volume celular. Src, EGFR e MAPK participam de maneira diferenciada na modulação destes efeitos. Observamos também que a radiação aumenta a motilidade dessas células via Src e MAPK e não induz alteração na proliferação celular até 48 horas após o tratamento. Este é o primeiro trabalho que correlaciona vias de sobrevivência celular como Src, EGFR e MAPK com alterações nas proteínas de junção aderente, alterações do citoesqueleto e migração celular. Estes eventos são relacionados aos efeitos colaterais primários e tardios induzidos pela IR, e podem favorecer à aquisição de um fenótipo maligno herdável durante o fracionamento de doses na RT, favorecendo a progressão tumoral do câncer colo-retal. Logo, além da correlação das vias de sinalização envolvidas nos eventos induzidos pela IR mostrados neste estudo, os resultados também corroboram para um melhor entendimento da atividade farmacológica dos inibidores químicos utilizados, uma vez que muitos deles encontram-se em fase de ensaios pré-clínicos e clínicos.

白杄花粉管微丝骨架介导信号传导的蛋白质组学研究

在高等植物有性生殖过程中，花粉管作为运输精子到胚珠的载体，它的生长具有高度极性化，并且要依赖于微丝。由于花粉管本身所具的特性，它已经成为研究细胞相互识别、胞内和胞外信号的模式系统。本文为了研究微丝在白杄（Picea meyeri Rehd. et Wils.）花粉管生长中的作用，我们应用不同浓度的微丝聚合抑制剂Latrunculin B (LATB) 处理花粉管，并通过激光共聚焦显微镜观察微丝聚合状态的动态变化。结果发现，在低浓度下的LATB能使花粉管中的微丝严重解聚，且抑制其顶端生长。 我们进一步利用蛋白质组学的手段，分析了白杄花粉管微丝解聚后蛋白质的表达图谱。通过双向电泳分离出500个左右考马斯亮兰染色的蛋白质斑点，经过软件分析发现，其中大部分蛋白质的表达量未发生变化，而只有110个蛋白斑点有较大变化。将这些蛋白斑点从胶上切下酶解后用于质谱鉴定，最终鉴定出35个蛋白，其中有18个为上调蛋白，17个下调蛋白。根据其主要功能，通常可分为碳水化合物代谢、胁迫反应、信号和细胞扩展等几类。我们发现由于微丝解聚引起的能量代谢水平降低，可能与依赖于信号传导的微丝重组过程相关。此外，当LATB浓度增加到50 nM时，与细胞壁多糖合成相关的两个蛋白，如reversibly glycosylated polypeptide和type IIIa membrane protein cp-wap13几乎不表达，这说明当微丝聚合完全被抑制后，依赖于微丝的分泌系统也受到影响，从而引起相应蛋白质变化，最终导致细胞壁成分合成的减少。细胞骨架蛋白actin的下调，进一步说明微丝在花粉管生长过程中起着提供或支持的一种机制，也就是能调节信号介导的花粉管生长，并使其在特定的时期到达特定的部位，从而完成植物的受精作用。

Characterizing the mechanics of cultured cell monolayers.

One-cell-thick monolayers are the simplest tissues in multicellular organisms, yet they fulfill critical roles in development and normal physiology. In early development, embryonic morphogenesis results largely from monolayer rearrangement and deformation due to internally generated forces. Later, monolayers act as physical barriers separating the internal environment from the exterior and must withstand externally applied forces. Though resisting and generating mechanical forces is an essential part of monolayer function, simple experimental methods to characterize monolayer mechanical properties are lacking. Here, we describe a system for tensile testing of freely suspended cultured monolayers that enables the examination of their mechanical behavior at multi-, uni-, and subcellular scales. Using this system, we provide measurements of monolayer elasticity and show that this is two orders of magnitude larger than the elasticity of their isolated cellular components. Monolayers could withstand more than a doubling in length before failing through rupture of intercellular junctions. Measurement of stress at fracture enabled a first estimation of the average force needed to separate cells within truly mature monolayers, approximately ninefold larger than measured in pairs of isolated cells. As in single cells, monolayer mechanical properties were strongly dependent on the integrity of the actin cytoskeleton, myosin, and intercellular adhesions interfacing adjacent cells. High magnification imaging revealed that keratin filaments became progressively stretched during extension, suggesting they participate in monolayer mechanics. This multiscale study of monolayer response to deformation enabled by our device provides the first quantitative investigation of the link between monolayer biology and mechanics.

Contractile and mechanical properties of epithelia with perturbed actomyosin dynamics.

Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. We combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. We found that a decrease in Myosin phosphorylation induces a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. We suggest that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through ECadherin-mediated adhesion. Altogether, our results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue.

Cortactin mediated morphogenic cell movements during zebrafish (Danio rerio) gastrulation

Cell migration is essential to direct embryonic cells to specific sites at which their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. To determine whether cortactin and Arp2/3 mediated actin assembly plays a role in the morphogenic cell movements during the early development of zebrafish, we initiated a study of cortactin expression in zebrafish embryos at gastrulating stages when massive cell migrations occur. Western blot analysis using a cortactin specific monoclonal antibody demonstrated that cortactin protein is abundantly present in embryos at the most early developmental stages. Immunostaining of whole-mounted embryo showed that cortactin immunoreactivity was associated with the embryonic shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining with cortactin and Arp3 antibodies also revealed that cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Therefore, our data suggest that cortactin and Arp2/3 mediated actin polymerization is implicated in the cell movement during gastrulation and perhaps the development of the central neural system as well.

Vaccinia virus assembly and motility

Vaccinia virus, the prototype member of the orthopoxviruses, is the largest and the most complex virus known. After replication of its genome and expression of the viral proteins, vaccinia undergoes a complicated assembly process which produces two distinct infectious forms. The first of these, the intracellular mature virus (IMV), develops from the immature virion (IV) after packaging of the genome and cleavage of the core proteins. During the transition of the IV to the IMV, a new core structure develops in the centre of the virion, concomitantly with the appearance of spike-like structures which extend between this core and the surrounding membranes of the IMV. I describe the characterization of p39 (gene A4L) which is hypothesized to be one component of these spikes. p39 is a core protein, but has strong associations with the membranes surrounding the IMV, possibly due to an interaction with p21 (A17L). Due to its location between the core and the membranes of the IMV, p39 is ideally situated to act as a matrix-like linker protein and may play a role in the formation of the core during the transition of the IV to the IMV. The IMV is subsequently wrapped by a membrane cisterna derived from the trans Golgi network, to form the intracellular enveloped virus (IEV). I show that the IEV can co-opt the actin cytoskeleton of the host cell in order to induce the formation of actin tails which extend from one side of the virion. These actin tails propel the virus particle, both intra- and intercellularly, at speeds of up to 2.8µm/min. On reaching the plasma membrane, the virus particles project out from the cell surface at the tip of virally induced microvilli. The outer membrane of the IEV is thought to fuse with the plasma membrane at the tip of these projections, thus exposing the second infectious form of vaccinia. This is thought to be the means by which the cell-associated enveloped virus is presented to neighbouring cells, thereby facilitating the direct cell-to-cell spread of virus particles.

Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells.

The growth of stem cells can be modulated by physical factors such as extracellular matrix nanotopography. We hypothesize that nanotopography modulates cell behavior by changing the integrin clustering and focal adhesion (FA) assembly, leading to changes in cytoskeletal organization and cell mechanical properties. Human mesenchymal stem cells (hMSCs) cultured on 350 nm gratings of tissue-culture polystyrene (TCPS) and polydimethylsiloxane (PDMS) showed decreased expression of integrin subunits alpha2, alpha , alpha V, beta2, beta 3 and beta 4 compared to the unpatterned controls. On gratings, the elongated hMSCs exhibited an aligned actin cytoskeleton, while on unpatterned controls, spreading cells showed a random but denser actin cytoskeleton network. Expression of cytoskeleton and FA components was also altered by the nanotopography as reflected in the mechanical properties measured by atomic force microscopy (AFM) indentation. On the rigid TCPS, hMSCs on gratings exhibited lower instantaneous and equilibrium Young's moduli and apparent viscosity. On the softer PDMS, the effects of nanotopography were not significant. However, hMSCs cultured on PDMS showed lower cell mechanical properties than those on TCPS, regardless of topography. These suggest that both nanotopography and substrate stiffness could be important in determining mechanical properties, while nanotopography may be more dominant in determining the organization of the cytoskeleton and FAs.

Pathogenicity of Salmonella: SopE-mediated membrane ruffling is independent of inositol phosphate signals

Studies [Zhou, D. Chen, L.-M. Hernandez, L. Shears, S.B. and Galán, J.E. (2001) A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host-cell actin cytoskeleton rearrangements and bacterial internalization. Mol. Microbiol. 39, 248-259] with engineered Salmonella mutants showed that deletion of SopE attenuated the pathogen's ability to deplete host-cell InsP5 and remodel the cytoskeleton. We pursued these observations: In SopE-transfected host-cells, membrane ruffling was induced, but SopE did not dephosphorylate InsP5, nor did it recruit PTEN (a cytosolic InsP5 phosphatase) for this task. However, PTEN strengthened SopE-mediated membrane ruffling. We conclude SopE promotes host-cell InsP5 hydrolysis only with the assistance of other Salmonella proteins. Our demonstration that Salmonella-mediated cytoskeletal modifications are independent of inositolphosphates will focus future studies on elucidating alternate pathogenic consequences of InsP5 metabolism, including ion channel conductance and apoptosis.

Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate.

Full-length transient receptor potential (TRP) cation channel TRPC4alpha and shorter TRPC4beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca(2+) influx. In common with other "classical" TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4alpha but not TRPC4beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP(2)). In contrast, several other phosphoinositides (PI), including PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), had no effect or even potentiated TRPC4alpha indicating that PIP(2) inhibits TRPC4alpha in a highly selective manner. We show that PIP(2) binds to the C terminus of TRPC4alpha but not that of TRPC4beta in vitro. Its inhibitory action was dependent on the association of TRPC4alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP(2) breakdown appears to be a required step in TRPC4alpha channel activation as PIP(2) depletion alone was insufficient for channel opening, which additionally required Ca(2+) and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP(2)/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist-activated cation channels in ileal myocytes.

Chemotaxis of macrophages is abolished in the Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is a rare disease characterized by microthrombocytopenia, eczema and immune deficiency. In this study a direct-viewing chemotaxis chamber was used to analyse chemotactic responses of WAS neutrophils and macrophages in stable linear concentration gradients. In five patients with classic WAS, chemotaxis of macrophages but not of neutrophils was found to be abolished, whereas the speed of random motility of both cell types was found to be indistinguishable from control cells. This supports the existence of an essential functional link, previously suggested by biochemical studies, between Cdc42, WAS protein (WASp) and the actin cytoskeleton in primary human macrophages. Moreover, these data suggest that Cdc42-WASp-mediated filopodial extension is a requirement for chemotaxis but not for chemokinesis in these cells. Abnormal directional cell motility of macrophages and related antigen-presenting cells may play a significant part in the immune deficiency and eczema of WAS.

Burkholderia cenocepacia Type VI Secretion System Mediates Escape of Type II Secreted Proteins into the Cytoplasm of Infected Macrophages

Burkholderia cenocepacia is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in bacteria-containing membrane vacuoles (BcCVs) resembling arrested autophagosomes. Intracellular bacteria stimulate IL-1ß secretion in a caspase-1-dependent manner and induce dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS) is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with B. cenocepacia employ the NLRP3 inflammasome to induce IL-1ß secretion and pyroptosis. Moreover, IL-1ß secretion by B. cenocepacia-infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS). We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1ß secretion in a T6SS dependent manner. ZmpA and ZmpB are also required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results uncover a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.

Pharmacogenomic Profiling and Pathway Analyses Identify MAPK-Dependent Migration as an Acute Response to SN38 in p53 Null and p53-Mutant Colorectal Cancer Cells.

The topoisomerase I inhibitor irinotecan is used to treat advanced colorectal cancer and has been shown to have p53-independent anticancer activity. The aim of this study was to identify the p53-independent signaling mechanisms activated by irinotecan. Transcriptional profiling of isogenic HCT116 p53 wild-type and p53 null cells was carried out following treatment with the active metabolite of irinotecan, SN38. Unsupervised analysis methods showed that p53 status had a highly significant impact on gene expression changes in response to SN38. Pathway analysis indicated that pathways involved in cell motility [adherens junction, focal adhesion, mitogen-activated protein kinase (MAPK), and regulation of the actin cytoskeleton] were significantly activated in p53 null cells, but not p53 wild-type cells, following SN38 treatment. In functional assays, SN38 treatment increased the migratory potential of p53 null and p53-mutant colorectal cancer cell lines, but not p53 wild-type lines. Moreover, p53 null SN38-resistant cells were found to migrate at a faster rate than parental drug-sensitive p53 null cells, whereas p53 wild-type SN38-resistant cells failed to migrate. Notably, cotreatment with inhibitors of the MAPK pathway inhibited the increased migration observed following SN38 treatment in p53 null and p53-mutant cells. Thus, in the absence of wild-type p53, SN38 promotes migration of colorectal cancer cells, and inhibiting MAPK blocks this potentially prometastatic adaptive response to this anticancer drug.

Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up-regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF-beta function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP-2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C-zeta to the leading edge of migrating cells. Inhibition of CTGF-induced protein kinase C-zeta activity with a myristolated PKC-zeta inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC-zeta kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF-induced migration compared with wild-type. Furthermore, CTGF stimulated phosphorylation and activation of GSK-3beta. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC-zeta and GSK-3beta. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.