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      Search for inhibitors of endocytosis : Intended specificity and unintended consequences

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          Abstract

          We discuss here the variety of approaches that have been taken to inhibit different forms of endocytosis. Typically, both non-specific and specific chemical inhibitors of endocytosis are tried in order to “classify” entry of a new plasma membrane protein into one of the various types of endocytosis. This classification can be confirmed through genetic approaches of protein depletion or overexpression of mutants of known endocytosis machinery components. Although some new compounds have been designed to be selective in biochemical assays, we caution investigators to be alert to the unintended consequences that sometimes arise when these compounds are applied to intact cells.

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          Regulated portals of entry into the cell.

          The plasma membrane is the interface between cells and their harsh environment. Uptake of nutrients and all communication among cells and between cells and their environment occurs through this interface. 'Endocytosis' encompasses several diverse mechanisms by which cells internalize macromolecules and particles into transport vesicles derived from the plasma membrane. It controls entry into the cell and has a crucial role in development, the immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. As the complexity of molecular interactions governing endocytosis are revealed, it has become increasingly clear that it is tightly coordinated and coupled with overall cell physiology and thus, must be viewed in a broader context than simple vesicular trafficking.
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            Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation

            The clathrin-coated pit lattice is held onto the plasma membrane by an integral membrane protein that binds the clathrin AP-2 subunit with high affinity. In vitro studies have suggested that this protein controls the assembly of the pit because membrane bound AP-2 is required for lattice assembly. If so, the AP-2 binding site must be a resident protein of the coated pit and recycle with other receptors that enter cells through this pathway. Proper recycling, however, would require the switching off of AP-2 binding to allow the binding site to travel through the endocytic pathway unencumbered. Evidence for this hypothesis has been revealed by the cationic amphiphilic class of drugs (CAD), which have previously been found to inhibit receptor recycling. Incubation of human fibroblasts in the presence of these drugs caused clathrin lattices to assemble on endosomal membranes and at the same time prevented coated pit assembly at the cell surface. These effects suggest that CADs reverse an on/off switch that controls AP-2 binding to membranes. We conclude that cells have a mechanism for switching on and off AP-2 binding during the endocytic cycle.
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              Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles.

              The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-beta-cyclodextrin (MbetaCD) to selectively extract cholesterol from the plasma membrane. MbetaCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MbetaCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MbetaCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MbetaCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.

                Author and article information

                Journal
                Cell Logist
                Cell Logist
                CL
                Cellular Logistics
                Landes Bioscience
                2159-2780
                2159-2799
                01 October 2012
                01 October 2012
                : 2
                : 4
                : 203-208
                Affiliations
                Cell Biology & Physiology Center; National Heart, Lung, and Blood Institute; National Institutes of Health; Bethesda, MD USA
                Author notes
                [* ]Correspondence to: Julie G. Donaldson, Email: jdonalds@ 123456helix.nih.gov
                Article
                2012CELLULARLOG0030R 23967
                10.4161/cl.23967
                3607622
                23538558
                f79c0ed5-7a2f-4eda-af94-d32f0e91abcc
                Copyright © 2012 Landes Bioscience

                This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.

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                Cell biology
                chemical inhibitor,phagocytosis,clathrin-mediated endocytosis,clathrin-independent endocytosis,pinocytosis,inhibitor,endocytosis

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