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First published online May 14, 2007
doi: 10.1242/10.1242/jcs.000653

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EGF induces macropinocytosis and SNX1-modulated recycling of E-cadherin

David M. Bryant^1,*, Markus C. Kerr¹, Luke A. Hammond¹, Shannon R. Joseph¹, Keith E. Mostov², Rohan D. Teasdale¹ and Jennifer L. Stow^1,

¹ Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
² Department of Anatomy, University of California, San Francisco, CA 94143, USA

View larger version (57K): [in this window] [in a new window]   Fig. 1. EGF induces membrane ruffling and internalization of E-cadherin-GFP. (A) MCF-7 cells were fixed and labeled for F-actin using Alexa Fluor-488-conjugated Phalloidin either (a) without or (b) with prior EGF stimulation for 1 hour. Arrows indicate stimulation of actin-rich membrane ruffles in response to EGF. (B) MCF-7 cells stably overexpressing GFP-tagged E-cadherin (hE-GFP-MCF-7 cells) were imaged live every 12 seconds over a 1-hour treatment period with EGF using a TILLvision live imaging microscope. An example of E-cad-GFP-positive membrane ruffling is depicted in cropped movie frames 150-157 (30:00-31:24 minutes) of the boxed region of interest in a. Arrows depict ruffling membranes. See accompanying Movie 1 in supplementary material. (C) hE-GFP-MCF-7 cells were stimulated with EGF and imaged live. See accompanying Movie 2 in supplementary material. The diagram pinpoints the macropinocytosis event and cell features depicted in the movie and the subsequent intensity-coloured still frames show the main steps in the internalization of surface hE-cad-GFP (green arrow) into a macropinosome (white arrow) and its recycling (green arrowhead) back to the surface in a region of cell-cell contact. Bars, 20 µm for a and b; 2 µm for cropped movie frames.

View larger version (106K): [in this window] [in a new window]   Fig. 2. EGF induces macropinocytosis of E-cadherin. (A) MCF-7 cells were treated with EGF for 10 minutes, fixed and immunolabeled for endogenous E-cadherin with EEA1 (a) or EGFR-GFP (b). Nuclei were stained with DAPI. Arrows denote localization of E-cadherin (a) and EGFR-GFP (b) to macropinosomes, arrowheads denote membrane ruffles. (B) MCF-7 cells were transiently transfected with YFP-rabankyrin-5, fixed and immunolabeled for endogenous E-cadherin (red) either (a) without or (b) with prior EGF stimulation for 10 minutes, and examined by epifluorescence. Arrows denote overlap of staining between intracellular E-cadherin and YFP-rabankyrin-5. Note formation of numerous macropinosomes upon EGF stimulation of YFP-rabankyrin-5-expressing cells. Second, third and fourth panels are magnifications of boxed areas shown in respective first panels. Bars, 20 µm for all first panels; 2 µm for all other panels.

View larger version (77K): [in this window] [in a new window]   Fig. 3. Macropinocytosis of catenins in conjunction with E-cadherin. MCF-7 cells were treated with EGF for 10 minutes, fixed and immunolabeled for E-cadherin (green) and nuclei (DAPI; blue), and for either (a) p120ctn (red) or (b) ß-catenin (red). Note localization of a pool of p120ctn and ß-catenin to E-cadherin-positive macropinosomes (arrows). Second, third and fourth panels are magnifications of boxed areas shown in respective first panels. Bars, 20 µm for all first panels; 2 µm for all other panels.

View larger version (107K): [in this window] [in a new window]   Fig. 4. Rac1 modulates macropinocytosis of E-cadherin. (A) Cells were serum-starved overnight, stimulated with EGF for 10 minutes and lysed. Then, levels of Rac1-GTP in equivalent concentrations of cell lysate were determined by PAK assay and compared with total Rac1 levels. Change in Rac1-GTP levels was quantified by densitometry. (B) MCF-7 cells were transiently transfected with either (a) wt-Rac1-GFP, (b) CA-Rac1-GFP or (c) CA-Cdc42-GFP (all in green), fixed and stained for endogenous E-cadherin (red) and nuclei (DAPI, blue) before confocal microscopy analysis. Note localization of E-cadherin to enlarged endosomal compartments labeled for CA-Rac1-GFP in b (arrows). Second, third and fourth panels are magnifications of boxed areas shown in respective first panels. Bars, 20 µm for all first panels; 2 µm for all other panels.

View larger version (104K): [in this window] [in a new window]   Fig. 5. EGF induces trafficking of a pool of E-cadherin to the late endosome. MCF-7 cells were stimulated with EGF (30 minutes), fixed and immunolabeled for E-cadherin (green) and nuclei (DAPI; blue). Antibodies against (a) GM130, (b) Rab11, or (c) cathepsin D (all in red) were used. White arrows denote co-labeling of E-cadherin and cathepsin D. Cropped panels (fourth column of panels) depict boxed region of interest at higher magnification. Bars, 20 µm.

View larger version (89K): [in this window] [in a new window]   Fig. 6. SNX1 localizes to E-cadherin-positive endosomal membranes. MCF-7 cells were fixed and stained for E-cadherin (green), nuclei (DAPI, blue) and SNX1 (red), without (a) or with prior EGF stimulation for 10 minutes (b) or 30 minutes (c) before confocal microscopy analysis. Arrows denote co-labeling of E-cadherin and SNX proteins on small typical endosomal puncta, arrowheads indicate localization of SNX1 to E-cadherin-positive macropinosomes. Note tubular extensions from macropinosomes in (b). Second, third and fourth panels are magnifications of boxed areas shown in respective first panels. Bars, 20 µm for all first panels; 2 µm for all other panels.

View larger version (53K): [in this window] [in a new window]   Fig. 7. SNX1 modulates E-cadherin intracellular trafficking and turnover. (A) MCF-7 cells were treated with either SNX1 siRNA or scrambled siRNA for 72 hours, and blotted for E-cadherin, SNX1 and SNX2. Notice the marked depletion of SNX1 levels in SNX1-siRNA-treated cells. (B) SNX1-depleted cells were stimulated with EGF for 10 minutes, fixed and stained for SNX1 (red) and E-cadherin (green). Inset depicts higher magnification of boxed regions of interest. Notice formation of an E-cadherin-containing macropinosome (arrows) in SNX1-depleted cells. (C) Lysates of SNX1-depleted or scramble siRNA-treated (control) cell monolayers were taken at either steady-state (total cell lysate, TCL) or following incubation with EGF for 0, 20 or 240 minutes, followed by trypsin-mediated cleavage of cell surface proteins. Total SNX1 and GAPDH levels were analyzed to ensure SNX1 knockdown and equivalent protein loading, respectively. Histograms were constructed from densitometry of immunoblots of intracellular (i.e. trypsin-protected) protein at relevant EGF treatment time points, and expressed as the percentage of total cellular protein (from TCL; **P>0.001, n=3, independent experiments). Notice a 45% increase in the intracellular pool of E-cadherin at 30 minutes of EGF stimulation in SNX1-depleted cells, but not for EGFR or TfnR. (D) hECD1 antibody was bound to surface E-cadherin in (b) SNX1-depleted and (a) scramble-siRNA-treated (control), followed by incubation with EGF for 30 minutes (c,d). Surface-antibody labeling of E-cadherin was stripped by mild-acid wash (c,d). Notice the relative increase in intracellular juxtanuclear E-cadherin labeling in SNX1-depleted cells. (E) Surface proteins in SNX1-depleted or scramble-siRNA-treated cell monolayers were biotinylated, and E-cadherin levels were analyzed without activation of internalization (0 hours), or after 4 hours or 18 hours of EGF stimulation. Histograms represent immunoblot intensity over various conditions as a percentage of the total level in control cells (scramble siRNA, 0 hours; **P>0.001, n=3, independent experiments). Bars, 20 µm.

View larger version (79K): [in this window] [in a new window]   Fig. 8. SNX1 modulates E-cadherin recycling. (A) SNX1-depleted cells and scramble-siRNA-treated cells (control) were fixed and stained for SNX1 (red), E-cadherin (green) and nuclei (DAPI, blue) at steady-state (a,d), after chelation of extracellular Ca2+ (b,e) or following restoration of Ca2+ levels for 60 minutes (c,f). Notice the retarded reformation of cell-cell junctions and the intracellular accumulation of E-cadherin (arrows) in SNX1-depleted cells. Insets depict higher magnification images of boxed regions. (B) Surface proteins in SNX1-depleted cell monolayers or scramble-siRNA-treated cell monolayers were biotinylated before Ca2+ chelation (Surface, Sur.), after chelation (EDTA) and after restoration of Ca2+ levels for 60 minutes (+Ca2+), and levels of surface E-cadherin, EGFR and TfnR were analyzed. Bar, 20 µm.