latrunculin-b and wiskostatin

In murine macrophages stimulated with macrophage-colony-stimulating factor (M-CSF), signals essential to macropinosome formation are restricted to the domain of plasma membrane enclosed within cup-shaped, circular ruffles. Consistent with a role for these actin-rich structures in signal amplification, microscopic measures of Rac1 activity determined that disruption of actin polymerization by latrunculin B inhibited ruffling and the localized activation of Rac1 in response to M-CSF. To test the hypothesis that circular ruffles restrict the lateral diffusion of membrane proteins that are essential for signaling, we monitored diffusion of membrane-tethered, photoactivatable green fluorescent protein (PAGFP-MEM) in ruffling and non-ruffling regions of cells. Although diffusion within macropinocytic cups was not inhibited, circular ruffles retained photoactivated PAGFP-MEM inside cup domains. Confinement of membrane molecules by circular ruffles could explain how actin facilitates positive feedback amplification of Rac1 in these relatively large domains of the plasma membrane, thereby organizing the contractile activities that close macropinosomes.

Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Cell Membrane; Diffusion; Fluorescent Dyes; Fluorometry; Green Fluorescent Proteins; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Neuropeptides; Pinocytosis; Propanolamines; rac GTP-Binding Proteins; rac1 GTP-Binding Protein; Signal Transduction; Thiazolidines; Transfection

The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid turnover at the plasma membrane in various cell types. The ubiquitously expressed N-WASP promotes actin polymerization and regulates endocytic trafficking of other proteins in response to signaling molecules such as Rho-GTPases. In the present study we investigated the effects of wiskostatin, an N-WASP inhibitor, on the surface expression and activity of CFTR. We demonstrate, using surface biotinylation methods, that the steady-state surface CFTR pool in stably transfected BHK cells was dramatically decreased following wiskostatin treatment with a corresponding increase in the amount of intracellular CFTR. Similar effects were observed for latrunculin B, a specific actin-disrupting reagent. Both reagents strongly inhibited macroscopic CFTR-mediated Cl(-) currents in two cell types including HT29-Cl19A colonic epithelial cells. As previously reported, CFTR internalization from the cell surface was strongly inhibited by a cyclic-AMP cocktail. This effect of cyclic-AMP was only partially blunted in the presence of wiskostatin, which raises the possibility that these two factors modulate different steps in CFTR traffic. In kinetic studies wiskostatin appeared to accelerate the initial rate of CFTR endocytosis as well as inhibit its recycling back to the cell surface over longer time periods. Our studies implicate a role for N-WASP-mediated actin polymerization in regulating CFTR surface expression and channel activity.

Topics: Actins; Animals; Biotinylation; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Cell Membrane; Colon; Cricetinae; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Dose-Response Relationship, Drug; Endocytosis; Epithelial Cells; HT29 Cells; Humans; Ion Channel Gating; Kinetics; Models, Biological; Patch-Clamp Techniques; Propanolamines; Thiazolidines; Wiskott-Aldrich Syndrome Protein, Neuronal

Differentiation and polarization of epithelial cells depends on the formation of the apical junctional complex (AJC), which is composed of the tight junction (TJ) and the adherens junction (AJ). In this study, we investigated mechanisms of actin reorganization that drive the establishment of AJC. Using a calcium switch model, we observed that formation of the AJC in T84 intestinal epithelial cells began with the assembly of adherens-like junctions followed by the formation of TJs. Early adherens-like junctions and TJs readily incorporated exogenous G-actin and were disassembled by latrunculin B, thus indicating dependence on continuous actin polymerization. Both adherens-like junctions and TJs were enriched in actin-related protein 3 and neuronal Wiskott-Aldrich syndrome protein (N-WASP), and their assembly was prevented by the N-WASP inhibitor wiskostatin. In contrast, the formation of TJs, but not adherens-like junctions, was accompanied by recruitment of myosin II and was blocked by inhibition of myosin II with blebbistatin. In addition, blebbistatin inhibited the ability of epithelial cells to establish a columnar phenotype with proper apico-basal polarity. These findings suggest that actin polymerization directly mediates recruitment and maintenance of AJ/TJ proteins at intercellular contacts, whereas myosin II regulates cell polarization and correct positioning of the AJC within the plasma membrane.

Topics: Actins; Adherens Junctions; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Cell Line; Cell Polarity; Epithelial Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Intestinal Mucosa; Microvilli; Models, Biological; Molecular Motor Proteins; Myosin Type II; Polymers; Propanolamines; Thiazoles; Thiazolidines; Tight Junctions