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      Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling

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          Abstract

          Inhibitors of Na +/H + exchange proteins block macropinocytosis by lowering the pH near the plasma membrane, which in turn inhibits actin remodeling by Rho family GTPases.

          Abstract

          Macropinocytosis is differentiated from other types of endocytosis by its unique susceptibility to inhibitors of Na +/H + exchange. Yet, the functional relationship between Na +/H + exchange and macropinosome formation remains obscure. In A431 cells, stimulation by EGF simultaneously activated macropinocytosis and Na +/H + exchange, elevating cytosolic pH and stimulating Na + influx. Remarkably, although inhibition of Na +/H + exchange by amiloride or HOE-694 obliterated macropinocytosis, neither cytosolic alkalinization nor Na + influx were required. Instead, using novel probes of submembranous pH, we detected the accumulation of metabolically generated acid at sites of macropinocytosis, an effect counteracted by Na +/H + exchange and greatly magnified when amiloride or HOE-694 were present. The acidification observed in the presence of the inhibitors did not alter receptor engagement or phosphorylation, nor did it significantly depress phosphatidylinositol-3-kinase stimulation. However, activation of the GTPases that promote actin remodelling was found to be exquisitely sensitive to the submembranous pH. This sensitivity confers to macropinocytosis its unique susceptibility to inhibitors of Na +/H + exchange.

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          Most cited references 82

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          The phosphoinositide 3-kinase pathway.

           Lewis Cantley (2002)
          Phosphorylated lipids are produced at cellular membranes during signaling events and contribute to the recruitment and activation of various signaling components. The role of phosphoinositide 3-kinase (PI3K), which catalyzes the production of phosphatidylinositol-3,4,5-trisphosphate, in cell survival pathways; the regulation of gene expression and cell metabolism; and cytoskeletal rearrangements are highlighted. The PI3K pathway is implicated in human diseases including diabetes and cancer, and understanding the intricacies of this pathway may provide new avenues for therapuetic intervention.
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            Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.

            In neural systems, information is often carried by ensembles of cells rather than by individual units. Optical indicators provide a powerful means to reveal such distributed activity, particularly when protein-based and encodable in DNA: encodable probes can be introduced into cells, tissues, or transgenic organisms by genetic manipulation, selectively expressed in anatomically or functionally defined groups of cells, and, ideally, recorded in situ, without a requirement for exogenous cofactors. Here we describe sensors for secretion and neurotransmission that fulfil these criteria. We have developed pH-sensitive mutants of green fluorescent protein ('pHluorins') by structure-directed combinatorial mutagenesis, with the aim of exploiting the acidic pH inside secretory vesicles to monitor vesicle exocytosis and recycling. When linked to a vesicle membrane protein, pHluorins were sorted to secretory and synaptic vesicles and reported transmission at individual synaptic boutons, as well as secretion and fusion pore 'flicker' of single secretory granules.
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              Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products

              We have previously demonstrated that human peripheral blood low density mononuclear cells cultured in granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 develop into dendritic cells (DCs) that are extremely efficient in presenting soluble antigens to T cells. To identify the mechanisms responsible for efficient antigen capture, we studied the endocytic capacity of DCs using fluorescein isothiocyanate-dextran, horseradish peroxidase, and lucifer yellow. We found that DCs use two distinct mechanisms for antigen capture. The first is a high level of fluid phase uptake via macropinocytosis. In contrast to what has been found with other cell types, macropinocytosis in DCs is constitutive and allows continuous internalization of large volumes of fluid. The second mechanism of capture is mediated via the mannose receptor (MR), which is expressed at high levels on DCs. At low ligand concentrations, the MR can deliver a large number of ligands to the cell in successive rounds. Thus, while macropinocytosis endows DCs with a high capacity, nonsaturable mechanism for capture of any soluble antigen, the MR gives an extra capacity for antigen capture with some degree of selectivity for non-self molecules. In addition to their high endocytic capacity, DCs from GM-CSF + IL-4-dependent cultures are characterized by the presence of a large intracellular compartment that contains high levels of class II molecules, cathepsin D, and lysosomal- associated membrane protein-1, and is rapidly accessible to endocytic markers. We investigated whether the capacity of DCs to capture and process antigen could be modulated by exogenous stimuli. We found that DCs respond to tumor necrosis factor alpha, CD40 ligand, IL-1, and lipopolysaccharide with a coordinate series of changes that include downregulation of macropinocytosis and Fc receptors, disappearance of the class II compartment, and upregulation of adhesion and costimulatory molecules. These changes occur within 1-2 d and are irreversible, since neither pinocytosis nor the class II compartment are recovered when the maturation-inducing stimulus is removed. The specificity of the MR and the capacity to respond to inflammatory stimuli maximize the capacity of DCs to present infectious non-self antigens to T cells.
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                Author and article information

                Affiliations
                [1 ]Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
                [2 ]Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
                [3 ]Laboratory of Physiology, School of Food and Nutritional Sciences, University of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka 422-8526, Japan
                [4 ]Department of Biochemistry, University of Geneva - Sciences II 30, CH-1211 Geneva, Switzerland
                [5 ]Department of Pediatrics and Physiology and [6 ]Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
                Author notes
                Correspondence to Sergio Grinstein: sga@ 123456sickkids.ca
                Journal
                J Cell Biol
                J. Cell Biol
                jcb
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                22 February 2010
                : 188
                : 4
                : 547-563
                200908086
                10.1083/jcb.200908086
                2828922
                20156964
                © 2010 Koivusalo et al.

                This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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