QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 26 July 2005
doi: 10.1242/jcs.02495

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tamma, G. Articles by Valenti, G. Search for Related Content PubMed PubMed Citation Articles by Tamma, G. Articles by Valenti, G.

Actin remodeling requires ERM function to facilitate AQP2 apical targeting

¹ Dipartimento di Fisiologia Generale ed Ambientale, University of Bari, Via Amendola 165/A, 70126 Bari, Italy
² Forschungsinstitut für Molekulare Pharmakologie, 13125 Berlin, Germany
³ Freie Universität Berlin, Institut für Pharmakologie, 14195 Berlin, Germany

View larger version (51K): [in a new window]   Fig. 1. Immunolocalization of moesin in CD8 cells. (A) Cells were either left untreated (CTR) or stimulated with forskolin (FK). After fixation, cells were immunostained with anti-moesin antibody (1:100 dilution). Fluorescence was visualized by epifluorescence microscopy and the xz reconstructions were obtained by deconvolution using Autodeblur software (insets). In untreated cells, moesin was predominantly localized at cell adhesion sites. In FK-stimulated cells, moesin was mainly expressed at cell-surface structures resembling apical microvilli. Bar, 5 µm. (B) Moesin distribution in subcellular fractions. Equal amounts of protein (30 µg/lane) from particulate (P, 150,000 g pellet) and soluble (S, 150,000 g supernatant) fractions from control and forskolin-stimulated cells were separated by gel electrophoresis and immunoblotted with anti-moesin antibody. Equal loading was confirmed by Coomassie blue staining. On the right, the densitometric analysis of the revealed bands is shown (*P<0.01, #P<0.001, with respect to control; n=3).

View larger version (44K): [in a new window]   Fig. 2. (A) Primary structure of the ERM peptide which reproduces a short sequence located in the C-terminal region of the ERM proteins within the binding site for F-actin (Turunen et al., 1994). An irrelevant peptide having a reversed sequence was used as a control, the control peptide. (B) Fluorescence localization of the ERM peptide in CD8 cells (2 µM, for 3 hours). (C) Moesin distribution in the soluble fraction. Cells were left untreated either stimulated with forskolin or pretreated for 1 or 3 hours with the ERM peptide or preincubated with the control peptide. After homogenization, the amount of protein in the 150,000 g supernatant was determined. Equal amounts of protein (30 µg/lane) were separated by gel electrophoresis and immunoblotted with anti-moesin antibody. Equal loading was confirmed by Coomassie blue staining. Notably, preincubation with the ERM peptide was associated with a strong and time-dependent decrease in moesin abundance in the soluble fraction – an effect similar to, although more potent than, that observed with forskolin treatment.

View larger version (49K): [in a new window]   Fig. 3. Effect of the ERM peptide on actin organization. (A) Cells were either left untreated (CTR) or stimulated with forskolin (FK) with or without preincubation for 3 hours with the ERM peptide or with the control peptide. F-actin was stained with TRITC-conjugated phalloidin and visualized by epifluorescence microscopy. Bar, 5 µm. (B) F-actin quantization by actin polymerization assay. Confluent cells were either left untreated (CTR) or stimulated with forskolin (FK) or preincubated for 3 hours with the ERM peptide. As internal control, cells were incubated with a control peptide with a reversed sequence with respect to the ERM peptide. After staining with TRITC-phalloidin, cells were extracted with cold methanol and the fluorescence absorbance of extracts was read (540/565 nm) in a RF-5301PC fluorimeter. The values obtained were compared by a one-way Anova and Tukey's multiple comparison test (#P<0.001).

View larger version (62K): [in a new window]   Fig. 4. Immunolocalization of AQP2 in cells pretreated with the ERM peptide. (A) Cells were either left untreated or stimulated with forskolin in the presence or absence of either ERM peptide or the control peptide. Fluorescence was visualized by epifluorescence microscopy and the xz reconstructions were obtained by deconvolution using Autodeblur software (shown in the insets). Forskolin stimulation caused AQP2 translocation from an intracellular pool to the apical membrane. A similar effect was observed on preincubation with the ERM peptide, while control peptide had no effect on AQP2 cellular localization. (B) Ratios of cell membrane/intracellular membrane fluorescence signals. Signal immunofluorescence intensities were detected from deconvoluted cells (Autodeblur software). The intracellular and cell membrane immunofluorescence signal intensities were calculated by using Metamorph software and normalized to the background signal intensities (n=15 for the control, n=13 for forskolin, n=13 for peptide control, n=13 for peptide control in the presence of forskolin, n=15 for ERM peptide, n=15 for ERM peptide in the presence of forskolin). Ratios greater than 1 indicate a cell membrane localization of AQP2. (#P<0.001 with respect to control). Values are expressed as means ± s.e.

View larger version (56K): [in a new window]   Fig. 5. Moesin association with actin cytoskeleton. The association of moesin with actin cytoskeleton was determined by its solubility in Triton X-100, which preserves the cytoskeleton and the cytoskeleton-associated proteins. Confluent cells were either left untreated (CTR) or stimulated with forskolin (FK) or preincubated for 3 hours with the control peptide or with the ERM peptide or with Y27632, an inhibitor of Rho kinase. Cells were incubated in Triton X-100 buffer and extracted proteins were immunoblotted with moesin antibody (A) or with P-Thr-moesin antibodies (B); on the right of each panel, the densitometric analysis of the bands obtained was shown. Histograms show the ratios between the densitometric signals of P-Thr-moesin and total moesin in each Triton-soluble fraction (C). (*P<0.01, **P<0.05, #P<0.001). Actin depolymerization is accompanied by a dissociation of moesin from actin cytoskeleton with a consistent reduction in P-Thr-moesin abundance. (D) Immunolocalization of P-Thr-moesin in CD8 cells. Cells were either left untreated or stimulated with forskolin. Fluorescence was visualized by epifluorescence microscopy. In untreated cells, P-Thr-moesin was localized at the cell-cell adhesion sites and along intracellular filaments depicting actin stress fibers (arrowheads). This particular staining disappeared in forskolin-stimulated cells. Bars, 5 µm.