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    Abstract: The host cell restriction factor CD317/tetherin traps virions at the surface of producer cells to prevent their release. The HIV-1 accessory protein Vpu antagonizes this restriction. Vpu reduces the cell surface density of the restriction factor and targets it for degradation; however, these activities are dispensable for enhancing particle release. Instead, Vpu has been suggested to antagonize CD317/tetherin by preventing recycling of internalized CD317/tetherin to the cell surface, blocking anterograde transport of newly synthesized CD317/tetherin, and/or displacing the restriction factor from virus assembly sites at the plasma membrane. At the molecular level, antagonism relies on the physical interaction of Vpu with CD317/tetherin. Recent findings suggested that phosphorylation of a diserine motif enables Vpu to bind to adaptor protein 1 (AP-1) trafficking complexes via two independent interaction motifs and to couple CD317/tetherin to the endocytic machinery. Here, we used a panel of Vpu proteins with specific mutations in individual interaction motifs to define which interactions are required for antagonism of CD317/tetherin. Impairing recycling or anterograde transport of CD317/tetherin to the plasma membrane was insufficient for antagonism. In contrast, excluding CD317/tetherin from HIV-1 assembly sites depended on Vpu motifs for interaction with AP-1 and CD317/tetherin and correlated with antagonism of the particle release restriction. Consistently, interference with AP-1 function or its expression blocked these Vpu activities. Our results define displacement from HIV-1 assembly sites as active principle of CD317/tetherin antagonism by Vpu and support a role of tripartite complexes between Vpu, AP-1, and CD317/tetherin in this process. IMPORTANCE: CD317/tetherin poses an intrinsic barrier to human immunodeficiency virus type 1 (HIV-1) replication in human cells by trapping virus particles at the surface of producer cells and thereby preventing their release. The viral protein Vpu antagonizes this restriction, and molecular interactions with the restriction factor and adaptor protein complex 1 (AP-1) were suggested to mediate this activity. Vpu modulates intracellular trafficking of CD317/tetherin and excludes the restriction factor from HIV-1 assembly sites at the plasma membrane, but the relative contribution of these effects to antagonism remain elusive. Using a panel of Vpu mutants, as well as interference with AP-1 function and expression, we show here that Vpu antagonizes CD317/tetherin by blocking its recruitment to viral assembly sites in an AP-1-dependent manner. These results refine our understanding of the molecular mechanisms of CD317/tetherin antagonism and suggest complexes of Vpu with the restriction factor and AP-1 as targets for potential therapeutic intervention.
    Type of Publication: Journal article published
    PubMed ID: 27170757
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  • 3
    Abstract: Essential characteristics of cellular signaling networks include a complex interconnected architecture and temporal dynamics of protein activity. The latter can be monitored by Forster resonance energy transfer (FRET) biosensors at a single-live-cell level with high temporal resolution. However, these experiments are typically limited to the use of a couple of FRET biosensors. Here, we describe a FRET-based multi-parameter imaging platform (FMIP) that allows simultaneous high-throughput monitoring of multiple signaling pathways. We apply FMIP to monitor the crosstalk between epidermal growth factor receptor (EGFR) and insulin-like growth factor-1 receptor signaling, signaling perturbations caused by pathophysiologically relevant EGFR mutations, and the effects of a clinically important MEK inhibitor (selumetinib) on the EGFR network. We expect that in the future the platform will be applied to develop comprehensive models of signaling networks and will help to investigate the mechanism of action as well as side effects of therapeutic treatments.
    Type of Publication: Journal article published
    PubMed ID: 27939899
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  • 4
    Keywords: TRAFFICKING ; LOCALIZATION ; MAMMALIAN-CELLS ; REVEALS ; ENDOPLASMIC-RETICULUM ; MOLECULAR-MECHANISMS ; GLOBAL ANALYSIS ; TO-GOLGI TRANSPORT ; COPII VESICLE FORMATION ; RAB GTPASE
    Abstract: The secretory pathway in mammalian cells has evolved to facilitate the transfer of cargo molecules to internal and cell surface membranes. Use of automated microscopy-based genome-wide RNA interference screens in cultured human cells allowed us to identify 554 proteins influencing secretion. Cloning, fluorescent-tagging and subcellular localization analysis of 179 of these proteins revealed that more than two-thirds localize to either the cytoplasm or membranes of the secretory and endocytic pathways. The depletion of 143 of them resulted in perturbations in the organization of the COPII and/or COPI vesicular coat complexes of the early secretory pathway, or the morphology of the Golgi complex. Network analyses revealed a so far unappreciated link between early secretory pathway function, small GTP-binding protein regulation, actin cytoskeleton organization and EGF-receptor-mediated signalling. This work provides an important resource for an integrative understanding of global cellular organization and regulation of the secretory pathway in mammalian cells.
    Type of Publication: Journal article published
    PubMed ID: 22660414
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  • 5
    Keywords: CELLS ; IN-VITRO ; CELL ; Germany ; screening ; SYSTEM ; GENES ; PROTEIN ; PROTEINS ; DIFFERENTIATION ; COMPLEX ; COMPLEXES ; LOCALIZATION ; cytoskeleton ; MICROTUBULES ; LIVING CELLS ; REGULATOR ; NEURONS ; ACTIN ; ELONGATION ; ADENOMATOUS POLYPOSIS-COLI ; NERVE GROWTH-FACTOR
    Abstract: Neurons, with their long axons and elaborate dendritic arbour, establish the complex circuitry that is essential for the proper functioning of the nervous system. Whereas a catalogue of structural, molecular, and functional differences between axons and dendrites is accumulating, the mechanisms involved in early events of neuronal differentiation, such as neurite initiation and elongation, are less well understood, mainly because the key molecules involved remain elusive. Here we describe the establishment and application of a microscopy-based approach designed to identify novel proteins involved in neurite initiation and/or elongation. We identified 21 proteins that affected neurite outgrowth when ectopically expressed in cells. Complementary time-lapse microscopy allowed us to discriminate between early and late effector proteins. Localization experiments with GFP-tagged proteins in fixed and living cells revealed a further 14 proteins that associated with neurite tips either early or late during neurite outgrowth. Coexpression experiments of the new effector proteins provide a first glimpse on a possible functional relationship of these proteins during neurite outgrowth. Altogether, we demonstrate the potential of the systematic microscope-based screening approaches described here to tackle the complex biological process of neurite outgrowth regulation
    Type of Publication: Journal article published
    PubMed ID: 17093056
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