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PubMed Central (PMC3 - NLM DTD) (2.987.316 recursos)

Archive of life sciences journal literature at the U.S. National Institutes of Health (NIH), developed and managed by NIH's National Center for Biotechnology Information (NCBI) in the National Library of Medicine (NLM).

Mostrando recursos 1 - 20 de 1.222

  1. Akt signaling dynamics in individual cells

    Gross, Sean M.; Rotwein, Peter
    The protein kinase Akt (for which there are three isoforms) is a key intracellular mediator of many biological processes, yet knowledge of Akt signaling dynamics is limited. Here, we have constructed a fluorescent reporter molecule in a lentiviral delivery system to assess Akt kinase activity at the single cell level. The reporter, a fusion between a modified FoxO1 transcription factor and clover, a green fluorescent protein, rapidly translocates from the nucleus to the cytoplasm in response to Akt stimulation. Because of its long half-life and the intensity of clover fluorescence, the sensor provides a robust readout that can be tracked...

  2. Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis

    Hornik, Tamara C.; Vilalta, Anna; Brown, Guy C.
    Some apoptotic processes, such as phosphatidylserine exposure, are potentially reversible and do not necessarily lead to cell death. However, phosphatidylserine exposure can induce phagocytosis of a cell, resulting in cell death by phagocytosis: phagoptosis. Phagoptosis of neurons by microglia might contribute to neuropathology, whereas phagoptosis of tumour cells by macrophages might limit cancer. Here, we examined the mechanisms by which BV-2 microglia killed co-cultured pheochromocytoma (PC12) cells that were either undifferentiated or differentiated into neuronal cells. We found that microglia activated by lipopolysaccharide rapidly phagocytosed PC12 cells. Activated microglia caused reversible phosphatidylserine exposure on and reversible caspase activation in PC12...

  3. Assembly and maintenance of the flagellum attachment zone filament in Trypanosoma brucei

    Zhou, Qing; Hu, Huiqing; He, Cynthia Y.; Li, Ziyin
    Adhesion of motile flagella to the cell body in Trypanosoma brucei requires a filamentous cytoskeletal structure termed the flagellum attachment zone (FAZ). Despite its essentiality, the complete molecular composition of the FAZ filament and its roles in FAZ filament assembly remain poorly understood. By localization-based screening, we here identified a new FAZ protein, which we called FAZ2. Knockdown of FAZ2 disrupted the FAZ filament, destabilized multiple FAZ filament proteins and caused a cytokinesis defect. We also showed that FAZ2 depletion destabilized another new FAZ filament protein and several flagellum and cytoskeleton proteins. Furthermore, we identified CC2D and KMP11 as FAZ2...

  4. CALHM1 ion channel elicits amyloid-β clearance by insulin-degrading enzyme in cell lines and in vivo in the mouse brain

    Vingtdeux, Valérie; Chandakkar, Pallavi; Zhao, Haitian; Blanc, Lionel; Ruiz, Santiago; Marambaud, Philippe
    Alzheimer's disease is characterized by amyloid-β (Aβ) peptide accumulation in the brain. CALHM1, a cell-surface Ca2+ channel expressed in brain neurons, has anti-amyloidogenic properties in cell cultures. Here, we show that CALHM1 controls Aβ levels in vivo in the mouse brain through a previously unrecognized mechanism of regulation of Aβ clearance. Using pharmacological and genetic approaches in cell lines, we found that CALHM1 ion permeability and extracellular Ca2+ were required for the Aβ-lowering effect of CALHM1. Aβ level reduction by CALHM1 could be explained by an increase in extracellular Aβ degradation by insulin-degrading enzyme (IDE), extracellular secretion of which was...

  5. Differing roles of pyruvate dehydrogenase kinases during mouse oocyte maturation

    Hou, Xiaojing; Zhang, Liang; Han, Longsen; Ge, Juan; Ma, Rujun; Zhang, Xuesen; Moley, Kelle; Schedl, Tim; Wang, Qiang
    Pyruvate dehydrogenase kinases (PDKs) modulate energy homeostasis in multiple tissues and cell types, under various nutrient conditions, through phosphorylation of the α subunit (PDHE1α, also known as PDHA1) of the pyruvate dehydrogenase (PDH) complex. However, the roles of PDKs in meiotic maturation are currently unknown. Here, by undertaking knockdown and overexpression analysis of PDK paralogs (PDK1–PDK4) in mouse oocytes, we established the site-specificity of PDKs towards the phosphorylation of three serine residues (Ser232, Ser293 and Ser300) on PDHE1α. We found that PDK3-mediated phosphorylation of Ser293-PDHE1α results in disruption of meiotic spindle morphology and chromosome alignment and decreased total ATP levels,...

  6. A role for F-BAR protein Rga7p during cytokinesis in S. pombe

    Arasada, Rajesh; Pollard, Thomas D.
    F-BAR proteins are known to participate in cytokinesis, but their mechanisms are not well understood. Here we investigated Rga7p, an Schizosaccharomyces pombe F-BAR protein with a RhoGAP domain. Localization of Rga7p to the cytokinetic cleavage furrow depends on its F-BAR domain, actin filaments, the formins Cdc12p and For3p, and the presence of a contractile ring. Rga7p is not required for the constriction of the contractile ring but does participate in the transport of a β-glucan synthetase (Bgs4p) from the late Golgi compartments to plasma membrane that is adjacent to the contractile ring. Cells without Rga7p moved Bgs4p normally from the...

  7. Epiboly generates the epidermal basal monolayer and spreads the nascent mammalian skin to enclose the embryonic body

    Panousopoulou, Eleni; Hobbs, Carl; Mason, Ivor; Green, Jeremy B. A.; Formstone, Caroline J.
    Epiboly is a morphogenetic process that is employed in the surface ectoderm of anamniotes during gastrulation to cover the entire embryo. We propose here that mammals also utilise this process to expand the epidermis and enclose the body cavity and spinal cord with a protective surface covering. Our data supports a model whereby epidermal spreading is driven by the primary establishment of the epidermal basal progenitor monolayer through radial cell intercalation of a multi-layered epithelium towards the basal lamina. By using a suspension organotypic culture strategy, we find that this process is fibronectin-dependent and autonomous to the skin. The radial...

  8. Ca2+ is a key factor in α-synuclein-induced neurotoxicity

    Angelova, Plamena R.; Ludtmann, Marthe H. R.; Horrocks, Mathew H.; Negoda, Alexander; Cremades, Nunilo; Klenerman, David; Dobson, Christopher M.; Wood, Nicholas W.; Pavlov, Evgeny V.; Gandhi, Sonia; Abramov, Andrey Y.
    Aggregation of α-synuclein leads to the formation of oligomeric intermediates that can interact with membranes to form pores. However, it is unknown how this leads to cell toxicity in Parkinson's disease. We investigated the species-specific effects of α-synuclein on Ca2+ signalling in primary neurons and astrocytes using live neuronal imaging and electrophysiology on artificial membranes. We demonstrate that α-synuclein induces an increase in basal intracellular Ca2+ in its unfolded monomeric state as well as in its oligomeric state. Electrophysiology of artificial membranes demonstrated that α-synuclein monomers induce irregular ionic currents, whereas α-synuclein oligomers induce rare discrete channel formation events. Despite...

  9. Screen-based identification and validation of four new ion channels as regulators of renal ciliogenesis

    Slaats, Gisela G.; Wheway, Gabrielle; Foletto, Veronica; Szymanska, Katarzyna; van Balkom, Bas W. M.; Logister, Ive; Den Ouden, Krista; Keijzer-Veen, Mandy G.; Lilien, Marc R.; Knoers, Nine V.; Johnson, Colin A.; Giles, Rachel H.
    To investigate the contribution of ion channels to ciliogenesis, we carried out a small interfering RNA (siRNA)-based reverse genetics screen of all ion channels in the mouse genome in murine inner medullary collecting duct kidney cells. This screen revealed four candidate ion channel genes: Kcnq1, Kcnj10, Kcnf1 and Clcn4. We show that these four ion channels localize to renal tubules, specifically to the base of primary cilia. We report that human KCNQ1 Long QT syndrome disease alleles regulate renal ciliogenesis; KCNQ1-p.R518X, -p.A178T and -p.K362R could not rescue ciliogenesis after Kcnq1-siRNA-mediated depletion in contrast to wild-type KCNQ1 and benign KCNQ1-p.R518Q, suggesting...

  10. Rab32 is essential for maintaining functional acidocalcisomes, and for growth and infectivity of Trypanosoma cruzi

    Niyogi, Sayantanee; Jimenez, Veronica; Girard-Dias, Wendell; de Souza, Wanderley; Miranda, Kildare; Docampo, Roberto
    The contractile vacuole complex (CVC) of Trypanosoma cruzi, the etiologic agent of Chagas disease, collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress; it also has a role in cell shrinking after hyperosmotic stress. Here, we report that, in addition to its role in osmoregulation, the CVC of T. cruzi has a role in the biogenesis of acidocalcisomes. Expression of dominant-negative mutants of the CVC-located small GTPase Rab32 (TcCLB.506289.80) results in lower numbers of less-electron-dense acidocalcisomes, lower content of polyphosphate, lower capacity for acidocalcisome acidification and Ca2+ uptake that is driven by the vacuolar...

  11. SEA you later alli-GATOR – a dynamic regulator of the TORC1 stress response pathway

    Dokudovskaya, Svetlana; Rout, Michael P.
    Cells constantly adapt to various environmental changes and stresses. The way in which nutrient and stress levels in a cell feed back to control metabolism and growth are, unsurprisingly, extremely complex, as responding with great sensitivity and speed to the ‘feast or famine, slack or stress’ status of its environment is a central goal for any organism. The highly conserved target of rapamycin complex 1 (TORC1) controls eukaryotic cell growth and response to a variety of signals, including nutrients, hormones and stresses, and plays the key role in the regulation of autophagy. A lot of attention has been paid recently...

  12. Autocrine VEGF maintains endothelial survival through regulation of metabolism and autophagy

    Domigan, Courtney K.; Warren, Carmen M.; Antanesian, Vaspour; Happel, Katharina; Ziyad, Safiyyah; Lee, Sunyoung; Krall, Abigail; Duan, Lewei; Torres-Collado, Antoni X.; Castellani, Lawrence W.; Elashoff, David; Christofk, Heather R.; van der Bliek, Alexander M.; Potente, Michael; Iruela-Arispe, M. Luisa
    Autocrine VEGF is necessary for endothelial survival, although the cellular mechanisms supporting this function are unknown. Here, we show that – even after full differentiation and maturation – continuous expression of VEGF by endothelial cells is needed to sustain vascular integrity and cellular viability. Depletion of VEGF from the endothelium results in mitochondria fragmentation and suppression of glucose metabolism, leading to increased autophagy that contributes to cell death. Gene-expression profiling showed that endothelial VEGF contributes to the regulation of cell cycle and mitochondrial gene clusters, as well as several – but not all – targets of the transcription factor FOXO1....

  13. Inhibiting IκBβ–NFκB signaling attenuates the expression of select pro-inflammatory genes

    McKenna, Sarah; Wright, Clyde J.
    Multiple mediators of septic shock are regulated by the transcription factor nuclear factor κB (NFκB). However, complete NFκB inhibition can exacerbate disease, necessitating evaluation of targeted strategies to attenuate the pro-inflammatory response. Here, we demonstrate that in murine macrophages, low-dose NFκB inhibitors specifically attenuates lipopolysaccharide (LPS)-induced IκBβ degradation and the expression of a select subset of target genes (encoding IL1β, IL6, IL12β). Gain- and loss-of-function experiments demonstrate the necessary and sufficient role of inhibitor of NFκB family member IκBβ (also known as NFKBIB) in the expression of these genes. Furthermore, both fibroblasts and macrophages isolated from IκBβ overexpressing mice demonstrate...

  14. Regulation of Cdc42 polarization by the Rsr1 GTPase and Rga1, a Cdc42 GTPase-activating protein, in budding yeast

    Lee, Mid Eum; Lo, Wing-Cheong; Miller, Kristi E.; Chou, Ching-Shan; Park, Hay-Oak
    Cdc42 plays a central role in establishing polarity in yeast and animals, yet how polarization of Cdc42 is achieved in response to spatial cues is poorly understood. Using live-cell imaging, we found distinct dynamics of Cdc42 polarization in haploid budding yeast in correlation with two temporal steps of the G1 phase. The position at which the Cdc42–GTP cluster develops changes rapidly around the division site during the first step but becomes stabilized in the second step, suggesting that an axis of polarized growth is determined in mid G1. Cdc42 polarization in the first step and its proper positioning depend on...

  15. Induction of stable ER–plasma-membrane junctions by Kv2.1 potassium channels

    Fox, Philip D.; Haberkorn, Christopher J.; Akin, Elizabeth J.; Seel, Peter J.; Krapf, Diego; Tamkun, Michael M.
    Junctions between cortical endoplasmic reticulum (cER) and the plasma membrane are a subtle but ubiquitous feature in mammalian cells; however, very little is known about the functions and molecular interactions that are associated with neuronal ER–plasma-membrane junctions. Here, we report that Kv2.1 (also known as KCNB1), the primary delayed-rectifier K+ channel in the mammalian brain, induces the formation of ER–plasma-membrane junctions. Kv2.1 localizes to dense, cell-surface clusters that contain non-conducting channels, indicating that they have a function that is unrelated to membrane-potential regulation. Accordingly, Kv2.1 clusters function as membrane-trafficking hubs, providing platforms for delivery and retrieval of multiple membrane proteins....

  16. Rab11a regulates syntaxin 3 localization and microvillus assembly in enterocytes

    Knowles, Byron C.; Weis, Victoria G.; Yu, Shiyan; Roland, Joseph T.; Williams, Janice A.; Alvarado, Gabriela S.; Lapierre, Lynne A.; Shub, Mitchell D.; Gao, Nan; Goldenring, James R.
    Rab11a is a key component of the apical recycling endosome that aids in the trafficking of proteins to the luminal surface in polarized epithelial cells. Utilizing conditional Rab11a-knockout specific to intestinal epithelial cells, and human colonic epithelial CaCo2-BBE cells with stable Rab11a knockdown, we examined the molecular and pathological impact of Rab11a deficiency on the establishment of apical cell polarity and microvillus morphogenesis. We demonstrate that loss of Rab11a induced alterations in enterocyte polarity, shortened microvillar length and affected the formation of microvilli along the lateral membranes. Rab11a deficiency in enterocytes altered the apical localization of syntaxin 3. These data...

  17. DNA damage response clamp 9-1-1 promotes assembly of ZMM proteins for formation of crossovers and synaptonemal complex

    Shinohara, Miki; Hayashihara, Kayoko; Grubb, Jennifer T.; Bishop, Douglas K.; Shinohara, Akira
    Formation of crossovers between homologous chromosomes during meiosis is positively regulated by the ZMM proteins (also known as SIC proteins). DNA damage checkpoint proteins also promote efficient formation of interhomolog crossovers. Here, we examined, in budding yeast, the meiotic role of the heterotrimeric DNA damage response clamp composed of Rad17, Ddc1 and Mec3 (known as ‘9-1-1’ in other organisms) and a component of the clamp loader, Rad24 (known as Rad17 in other organisms). Cytological analysis indicated that the 9-1-1 clamp and its loader are not required for the chromosomal loading of RecA homologs Rad51 or Dmc1, but are necessary for...

  18. Hsp90α and Hsp90β together operate a hypoxia and nutrient paucity stress-response mechanism during wound healing

    Jayaprakash, Priyamvada; Dong, Hangming; Zou, Mengchen; Bhatia, Ayesha; O'Brien, Kathryn; Chen, Mei; Woodley, David T.; Li, Wei
    When tissues are injured and blood vessels clot, the local environment becomes ischemic, meaning that there is a lack of adequate supply of oxygen and glucose delivered to the surrounding cells. The heat shock protein-90 (Hsp90) family proteins protect tissues from various environmental insults and participate in the repair of damaged tissue. Here, we report discovery of a new ischemia-responsive mechanism in which the two Hsp90 isoforms Hsp90α and Hsp90β (also known as HSP90AA1 and HSP90AB1, respectively) work together to promote cell motility in wounded skin and accelerate wound closure. We demonstrate that Hsp90α and Hsp90β have distinct and non-exchangeable...

  19. MicroRNA-mediated regulation of p21 and TASK1 cellular restriction factors enhances HIV-1 infection

    Farberov, Luba; Herzig, Eytan; Modai, Shira; Isakov, Ofer; Hizi, Amnon; Shomron, Noam
    MicroRNAs (miRNAs) are short non-coding RNAs that play a central role in the regulation of gene expression by binding to target mRNAs. Several studies have revealed alterations in cellular miRNA profiles following HIV-1 infection, mostly for miRNAs involved in inhibiting viral infection. These miRNA expression modifications might also serve to block the innate HIV-1 inhibition mechanism. As a result, it is expected that during HIV-1 infection miRNAs target genes that hinder or prevent the progression of the HIV-1 replication cycle. One of the major sets of genes known to inhibit the progression of HIV-1 infection are cellular restriction factors. In...

  20. The golgin GMAP-210 is required for efficient membrane trafficking in the early secretory pathway

    Roboti, Peristera; Sato, Keisuke; Lowe, Martin
    Golgins are coiled-coil proteins that participate in membrane-tethering events at the Golgi complex. Golgin-mediated tethering is thought to be important for vesicular trafficking and Golgi organization. However, the degree to which individual golgins contribute to these processes is poorly defined, and it has been proposed that golgins act in a largely redundant manner. Previous studies on the golgin GMAP-210 (also known as TRIP11), which is mutated in the rare skeletal disorder achondrogenesis type 1A, have yielded conflicting results regarding its involvement in trafficking. Here, we re-investigated the trafficking role of GMAP-210, and found that it is indeed required for efficient...

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