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EGF Induces Macropinocytosis and SNX1-Modulated Recycling of E-Cadherin

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EGF Induces Macropinocytosis and SNX1-Modulated Recycling of E-Cadherin 1818 Research Article EGF induces macropinocytosis and SNX1-modulated recycling of E-cadherin David M. Bryant1,*, Markus C. Kerr1, Luke A. Hammond1, Shannon R. Joseph1, Keith E. Mostov2, Rohan D. Teasdale1 and Jennifer L. Stow1,‡ 1Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia 2Department of Anatomy, University of California, San Francisco, CA 94143, USA *Present address: Department of Anatomy, University of California, San Francisco, CA 94143, USA ‡Author for correspondence (e-mail: [email protected]) Accepted 5 April 2007 Journal of Cell Science 120, 1818-1828 Published by The Company of Biologists 2007 doi:10.1242/jcs.000653 Summary In epithelia, junction proteins are endocytosed for surface in response to EGF. Moreover, SNX1 was also modulation of cell-cell adhesion and cell polarity. In required for efficient recycling of internalized E-cadherin response to growth factors, the cell-cell adhesion protein E- and re-establishment of epithelial adhesion. Together, these cadherin is internalized from the cell surface with findings demonstrate a role for SNX1 in retrieval of E- degradation or recycling as potential fates. However, the cadherin from a degradative endosomal pathway and in cellular machinery involved in cadherin internalization membrane trafficking pathways that regulate E-cadherin and recycling remains controversial. Here we investigated recycling. EGF-induced E-cadherin internalization. EGF stimulation of MCF-7 cells resulted in Rac1-modulated macropinocytosis of the E-cadherin-catenin complex into Supplementary material available online at endosomal compartments that colocalized with EEA1 and http://jcs.biologists.org/cgi/content/full/120/10/1818/DC1 the sorting nexin, SNX1. Depletion of cellular SNX1 levels by siRNA resulted in increased intracellular accumulation Key words: Sorting Nexin, EGF, Cadherin, Macropinocytosis, and turnover of E-cadherin internalized from the cell Recycling, Cell-cell adhesion Introduction catenin proteins, ␤-catenin or ␥-catenin (plakoglobin), and ␤ Journal of Cell Science For epithelial cells in sheets or tubes, alteration of stable cell- p120ctn (Yap, 1998). Through the interaction of -catenin with cell adhesion facilitates morphological remodeling events, ␣-catenin, E-cadherin dynamically associates with cortical such as migration during development or in metastasis during actin, although the molecular details of this interaction have cancer (Gumbiner, 2005; Thiery, 2002). Internalization of recently become controversial (Drees et al., 2005; Yamada et junction proteins is one mechanism for temporary or al., 2005). In mammalian cells, binding of p120ctn to the permanent modulation of cell-cell adhesion (reviewed in juxtamembrane cytoplasmic region of E-cadherin delimits Bryant and Stow, 2004; D’Souza-Schorey, 2005; Ivanov et al., cadherin endocytosis and turnover, thereby stabilizing 2005). A number of cell-cell junction proteins have been cadherin-based adhesion (Davis et al., 2003; Xiao et al., 2003; demonstrated to internalize in response to various Yap et al., 1998). physiological stimuli. For instance, autoantibodies against We have previously shown that one mechanism to regulate desmoglein-3 generated in pemphigus disorders induce E-cadherin function at cell-cell contacts is to internalize this desmosome disassembly and internalization in epidermal cells adhesion receptor, with the potential for its recycling to the cell (Calkins et al., 2006), whereas IFN␥ stimulation of intestinal surface (Le et al., 2002; Le et al., 1999). FGF stimulation of epithelial sheets increases barrier permeability by temporarily mammary epithelial cells (MCF-7 cells) causes E-cadherin internalizing and recycling tight-junction proteins, such as endocytosis as a precursor to subsequent disruption of cell-cell JAM-A and occludin (Bruewer et al., 2005; Utech et al., 2005). adhesion (Bryant et al., 2005). In recent years, a number of In canine kidney epithelial cells, the adhesion protein E- molecular regulators of cadherin endocytosis have been cadherin undergoes constitutive endocytosis and recycling, and identified, including some in clathrin-dependent and clathrin- it is also internalized in response to hepatocyte growth factor independent pathways (Bryant and Stow, 2004; Le et al., 1999; (HGF) for modulation of cell-cell adhesion (Fujita et al., 2002; Palacios et al., 2002; Paterson et al., 2003), and a caveolin-1- Le et al., 1999). associated pathway (Lu et al., 2003). Endocytosed E-cadherin E-cadherin is the prototypical epithelial cadherin and, at can either be degraded or recycled to the cell surface – either steady-state, it is localized to the lateral surface of polarized mechanism leading to long-term or short-term modulation of epithelia, where homodimers interact in trans with E-cadherin cell-cell adhesion. What regulates the fate of cadherin once it dimers on apposing cell membranes (reviewed in Yap et al., is internalized from the cell surface is not known. 1997). The cytoplasmic domain of E-cadherin supports One potential set of candidates for regulation of E-cadherin interactions with multiple binding partners, most notably the intracellular traffic is the sorting nexins (SNXs), a family E-cadherin macropinocytosis and recycling 1819 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 ␮m for a and b; 2 ␮m for cropped movie frames. Journal of Cell Science of phosphatidylinositol 3-phosphate [PtdIns(3)P]-binding to function in the recycling of internalized E-cadherin back to proteins defined by a common PX domain, with varied roles in the cell surface and in the re-establishment of epithelial intracellular trafficking (Carlton et al., 2005; Worby and Dixon, polarity. 2002). SNX1 was originally identified as a binding partner and regulator of epidermal growth factor receptor (EGFR) Results trafficking and degradation (Kurten et al., 1996). More recent EGF induces membrane ruffling and macropinocytosis studies have assigned a function for SNX1, in conjunction with of the E-cadherin-catenin complex the mammalian retromer complex, in retrieval of internalized EGF stimulation can induce pleiotropic effects on epithelial proteins, such as the cation-independent mannose 6-phosphate cells, including proliferation, migration, invasion and receptor (CI-M6PR), away from the degradative pathway transformation (for a review, see Thiery and Chopin, 1999). (reviewed in Carlton et al., 2005). Different SNX proteins have We first examined the morphological consequence of EGFR now emerged as participating in alternate endosomal membrane activation in our system by stimulating poorly motile human sorting events, particularly in the traffic of internalized proteins breast adenocarcinoma (MCF-7) cells with EGF. Cell shape into and out of degradative endosomal pathway compartments was assessed by F-actin labeling. Cells grown in serum- (see above) (Carlton et al., 2005; Carlton and Cullen, 2005; Xu supplemented medium displayed F-actin labeling mainly at et al., 2001). cell-cell junctions, but also at surface membrane ruffles (Fig. We here set out to examine EGF-induced internalization of 1A). EGF induced prominent ruffling of the cell surface E-cadherin and the potential role of SNX1 in the intracellular membranes not in contact with other cells but did not result in trafficking of E-cadherin in MCF-7 cells. Unlike the FGF- overt disruption of junctional labeling or cell-cell adhesion induced endocytosis of E-cadherin into early endosomes we (Fig. 1A). MCF-7 cells stably expressing E-cadherin-GFP (hE- previously described in these cells (Bryant et al., 2005), we GFP-MCF-7 cells) (Bryant et al., 2005) were examined by live here report EGF-induced, Rac1-modulated macropinocytosis cell imaging. E-cadherin-GFP was concentrated at cell-cell of E-cadherin, ␤-catenin and p120ctn. Interestingly, this occurs contacts and was also visible in intracellular vesicles (Fig. 1B), without downregulation of total E-cadherin levels, suggesting probably representing exocytic and recycling E-cadherin recycling of internalized E-cadherin. SNX1 localized to E- (Bryant and Stow, 2004). Similar to parental MCF-7 cells (Fig. cadherin-positive endocytic compartments and is shown here 1A), EGF stimulation caused ruffling of free cell surface 1820 Journal of Cell Science 120 (10) membranes whereas at other points cell-cell
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