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Publications (8)75.08 Total impact

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    ABSTRACT: Here we combined classical biochemistry with new biophysical approaches to study the organization of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) with high spatial and temporal resolution at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, after sorting in the Golgi, each GPI-AP reaches the apical surface in homoclusters. Golgi-derived homoclusters are required for their subsequent plasma membrane organization into cholesterol-dependent heteroclusters. By contrast, in nonpolarized MDCK cells, GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form heteroclusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, in contrast to fibroblasts, in polarized epithelial cells, a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and function of GPI-APs at the apical surface.
    Nature Chemical Biology 03/2014; · 12.95 Impact Factor
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    ABSTRACT: Angiogenin (ANG) promotes cell growth and survival. Under growth conditions, ANG undergoes nuclear translocation and is accumulated in nucleolus where it stimulates ribosomal RNA (rRNA) transcription. When cells are stressed, ANG mediates the production of tRNA-derived stress-induced small RNA (tiRNA) that reprograms protein translation into a survival mechanism. The ribonucleolytic activity of ANG is essential for both processes but how this activity is regulated is unknown. We report here that ribonuclease/angiogenin inhibitor1 (RNH1) controls both localization and activities of ANG. Under growth conditions, ANG is located in the nucleus and is not associated with RNH1 so that the ribonucleolytic activity is retained to ensure rRNA transcription, whereas cytoplasmic ANG is associated with and inhibited by RNH1 so that random cleavage of cellular RNA is prevented. Under stresses, ANG is located in cytoplasm and is concentrated in stress granules (SG) where it is not associated with RNH1 thus remains enzymatically active for tiRNA production. In contrast, nuclear ANG is associated with RNH1 in stressed cells to ensure that the enzymatic activity is inhibited and no unnecessary rRNA is produced to save anabolic energy. Knockdown of RNH1 abolished stress-induced relocalization of ANG and decreased cell growth and survival.
    Journal of Cell Science 07/2013; · 5.88 Impact Factor
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    ABSTRACT: Large pleiomorphic carriers leave the Golgi complex for the plasma membrane by en bloc extrusion of specialized tubular domains, which then undergo fission. Several components of the underlying molecular machinery have been identified, including those involved in the budding/initiation of tubular carrier precursors (for example, the phosphoinositide kinase PI(4)KIIIβ, the GTPase ARF, and FAPP2), and in the fission of these precursors (for example, PKD, CtBP1-S/BARS). However, how these proteins interact to bring about carrier formation is poorly understood. Here, we describe a protein complex that mediates carrier formation and contains budding and fission molecules, as well as other molecules, such as the adaptor protein 14-3-3γ. Specifically, we show that 14-3-3γ dimers bridge CtBP1-S/BARS with PI(4)KIIIβ, and that the resulting complex is stabilized by phosphorylation by PKD and PAK. Disrupting the association of these proteins inhibits the fission of elongating carrier precursors, indicating that this complex couples the carrier budding and fission processes.
    Nature Cell Biology 02/2012; 14(4):343-54. · 20.76 Impact Factor
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    ABSTRACT: Through negative regulation of gene expression, microRNAs (miRNAs) can function as oncosuppressors in cancers, and can themselves show altered expression in various tumor types. Here, we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many of the cell-fate-determining stages. Notch regulates a subset of MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulate these phenomena, and can be used in anti-cancer therapies. In a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1). Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Using an inducible tetracycline on-off model of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. MiR-34a expression negatively affects CD133(+)/CD15(+) tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal model of MB (Patch1(+/-) p53(-/-)), thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo. Despite advances in our understanding of the pathogenesis of MB, one-third of patients with MB remain incurable. Here, we show that stable nucleic-acid-lipid particles carrying mature miR-34a can target Dll1 in vitro and show equal effects to those of adenovirus miR-34a cell infection. Thus, this technology forms the basis for their therapeutic use for the delivery of miR-34a in brain-tumor treatment, with no signs of toxicity described to date in non-human primate trials.
    PLoS ONE 01/2011; 6(9):e24584. · 3.53 Impact Factor
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    ABSTRACT: Recently, Dawson et al identified a previously unrecognized nuclear role of JAK2 in the phosphorylation of histone H3 in hematopoietic cell lines. We searched nuclear JAK2 in total bone marrow (BM) cells and in 4 sorted BM cell populations (CD34(+), CD15(+), CD41(+), and CD71(+)) of 10 myeloproliferative neoplasia (MPN) patients with JAK2V617F mutation and 5 patients with wild-type JAK2 MPN. Confocal immunofluorescent images and Western blot analyses of nuclear and cytoplasmic fractions found nuclear JAK2 in CD34(+) cells of 10 of 10 JAK2-mutated patients but not in patients with wild-type JAK2. JAK2 was predominantly in the cytoplasmic fraction of differentiated granulocytic, megakaryocytic, or erythroid cells obtained from all patients. JAK2V617F up-regulates LMO2 in K562 and in JAK2V617F-positive CD34(+) cells. The selective JAK2 inhibitor AG490 normalizes the LMO2 levels in V617F-positive K562 and restores the cyto-plasmic localization of JAK2.
    Blood 12/2010; 116(26):6023-6. · 9.78 Impact Factor
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    ABSTRACT: Diatoms are an important group of eukaryotic phytoplankton, responsible for about 20% of global primary productivity. Study of the functional role of chemical signaling within phytoplankton assemblages is still in its infancy although recent reports in diatoms suggest the existence of chemical-based defense strategies. Here, we demonstrate how the accurate perception of diatom-derived reactive aldehydes can determine cell fate in diatoms. In particular, the aldehyde (2E,4E/Z)-decadienal (DD) can trigger intracellular calcium transients and the generation of nitric oxide (NO) by a calcium-dependent NO synthase-like activity, which results in cell death. However, pretreatment of cells with sublethal doses of aldehyde can induce resistance to subsequent lethal doses, which is reflected in an altered calcium signature and kinetics of NO production. We also present evidence for a DD-derived NO-based intercellular signaling system for the perception of stressed bystander cells. Based on these findings, we propose the existence of a sophisticated stress surveillance system in diatoms, which has important implications for understanding the cellular mechanisms responsible for acclimation versus death during phytoplankton bloom successions.
    PLoS Biology 04/2006; 4(3):e60. · 12.69 Impact Factor
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    ABSTRACT: Light provides a major source of information from the environment during plant growth and development. Recent results suggest that the key events controlling light-regulated gene expression in plants are translocation of the phytochrome photoreceptors into the nucleus, followed by their binding to transcription factors such as PIF3. Coupled with this, the degradation of positively acting intermediates such as the transcription factor HY5 by COP1 and the COP9 signalosome appears to be an important process whereby photomorphogenesis is repressed in darkness (e.g., ). Genetic analyses in Arabidopsis and tomato have revealed that the nuclear protein DET1 also plays a key role in the repression of photomorphogenesis. However, the function of this protein has remained a mystery. In a series of in vitro experiments, we provide persuasive evidence that DET1 binds to nonacetylated amino-terminal tails of the core histone H2B in the context of the nucleosome. Furthermore, we have utilized FRET (fluorescence resonance energy transfer) imaging with GFP variants to demonstrate this interaction within the nucleus of living plant cells. Given the dramatic photomorphogenic phenotypes of det1 mutants, we propose that chromatin remodeling plays a heretofore unsuspected role in regulating gene expression during photomorphogenesis.
    Current Biology 10/2002; 12(17):1529-34. · 9.49 Impact Factor
  • Fabio. Formiggini
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    ABSTRACT: Thesis (Ph.D.)--Open University, 2003.