1Institute for Molecular Bioscience and 2School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Queensland, Australia
Submitted 1 November 2004 ; accepted in final form 27 January 2005
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ABSTRACT |
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Rho family GTPases; catenin; polarity; sorting; actin
The dynamic trafficking of E-cadherin to and from the lateral surface of epithelial cells is essential to initially deliver newly synthesized E-cadherin to the adherens junction and thereafter to balance and modulate cadherin-based adhesion. It is now widely recognized that cell surface cadherins can be internalized constitutively and recycled in confluent epithelia or endocytosed in response to growth factors via different endocytic carriers and pathways (3, 33). The exocytosis of newly synthesized E-cadherin requires sorting and polarized transport to the basolateral membrane in epithelial cells. We have shown that this trafficking of E-cadherin is mediated by a dileucine motif in the juxtamembrane tail domain and that a mutant (E-cadS1) lacking this motif is mistargeted to the apical membrane in Madin-Darby canine kidney (MDCK) cells (36, 37). Other events documented during the exocytic trafficking of E-cadherin include the sequential assembly of the E-cadherin-catenin complex. While
-catenin appears to bind to E-cadherin early in the biosynthetic pathway, p120ctn binds to the complex more distally, at or near the basolateral membrane in MDCK cells (8, 36). The trafficking of N-cadherin involves earlier binding of p120ctn and an interaction via p120ctn with kinesin and microtubules during transport to the cell surface (7, 47).
Other regulatory molecules involved in the sorting or basolateral transport of E-cadherin have not yet been defined. However, relevant insights have emerged from studies on the trafficking of other basolateral membrane proteins. Some proteins that are sorted via tyrosine-based motifs for basolateral trafficking interact with a specific adaptor protein (AP) complex, AP-1B, of which the µ1B-subunit is expressed only in polarized epithelial cells (13). The exocyst is a multisubunit complex of vesicle and target membrane-associated proteins with essential roles in polarized secretion in yeast (22) and in basolateral exocytic trafficking in epithelial cells, where it defines the apicolateral point of delivery for vesicle carriers moving to the lateral cell membrane (20, 34). Other proteins that interact with the exocyst complex, including RalA (44) and Rho GTPases, also have been shown to regulate basolateral trafficking (35).
The Rho family proteins are monomeric G proteins that are involved in signaling pathways throughout eukaryotic cells and often control actin polymerization in diverse contexts (12). Two members of this family, Cdc42 and Rac1, often have complementary functions and common effectors, and both have been implicated in regulating exocytic and endocytic trafficking pathways (43). Cdc42 and Rac1 also contribute to the establishment of cell polarity as reported in developing wing epithelia in Drosophila (10). Multiple lines of evidence, including activation of endogenous Cdc42, show that Cdc42 is associated with membranes and vesicles at the trans-Golgi network (TGN) (11, 14, 40). In addition, Cdc42 participates in regulating post-Golgi trafficking to the basolateral cell surface in polarized epithelial cells (9, 32). Dominant-negative mutants of Cdc42 disrupt polarity in epithelial cells, sending basolateral proteins to the apical membrane (32).
On the basis of the known functions of Cdc42 and Rac1 in polarized protein trafficking, we set out to investigate specific roles for these GTPases in the basolateral trafficking of E-cadherin. An additional impetus for this study was the known function of Cdc42 and Rac1 in regulating the function and trafficking of E-cadherin once it reaches the cell surface. At the cell surface, RhoA, Rac1, and Cdc42 each act to directly regulate components of the cadherin-catenin complex to modulate cadherin-based adhesion and signaling (16). Rac1 has been shown to regulate the endocytosis of E-cadherin, specifically functioning to make nonadhesive E-cadherin available for internalization (27). While there is strong evidence linking Rho GTPases to E-cadherin at the cell surface, the participation of Cdc42 or Rac1 at an earlier stage during the biosynthesis or exocytic trafficking of E-cadherin has not been demonstrated. Our results show that both Rac1 and Cdc42 are required for the efficient post-Golgi sorting of E-cadherin and its delivery to the lateral cell surface. These findings show that Rac1 and Cdc42 are involved in determining the fate of E-cadherin at a much earlier stage than previously suspected.
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MATERIALS AND METHODS |
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Plasmids.
An EGFP vector (Clontech, Palo Alto, CA) encoding full-length human E-cadherin tagged with green fluorescent protein (GFP) at the COOH terminus was used as described previously (37). A mutant of this cDNA with the dileucine motif at amino acids 587 and 588 mutagenized to a dialanine motif (E-cadS1-GFP) also was used. This mutation results in the mistargeting of E-cadherin to the apical plasma membrane in polarized MDCK cells (36). Rac1 and Cdc42 cDNA were originally obtained from the laboratory of Dr. Alan Hall (Medical Research Council Laboratory of Cell Biology, London, UK). pcDNA3 expression vectors encoding wild-type Rac1 and Cdc42, dominant-active Rac1 (RacV12 mutation RacCA), constitutively active Cdc42 (Cdc42G12V mutation Cdc42CA) and dominant-negative forms of Rac1 (RacN17 mutation RacDN) and Cdc42 (Cdc42D57Y mutation Cdc42DN) tagged with c-myc were either transiently transfected or microinjected into MDCK cells. A pTet-Off plasmid (Clontech) was constructed to express tetracycline-inducible GFP and tetracycline-repressible c-myc-tagged Rac1DN bidirectionally and was kindly provided by Brooke Gardiner and Rick Sturm (University of Queensland, Brisbane, Australia). This plasmid was used to generate stably expressing MDCK cells.
Transfection. MDCK cells were plated at 2040% confluence onto glass coverslips 24 h before transfection with 2 µg of cDNA and Lipofectamine Plus, according to the manufacturer's instructions (GIBCO-BRL, San Diego, CA). Briefly, cells were transfected in serum-free media and incubated for 3 h at 37°C in 5% CO2, followed by the addition of an equal volume of normal medium containing 20% FCS for 1618 h (10% final serum concentration). The DNA-containing medium was then removed and replaced with standard growth medium. Cells were used for experiments 2448 h after transfection.
Microinjection.
Plasmids encoding cDNA of interest were diluted to a final concentration of 100 ng/µl in a microinjection buffer of 10 mM KH2PO4, pH 7.2, containing 75 mM KCl, and centrifuged for 15 min in a benchtop microfuge (Eppendorf, Hamburg, Germany) at 14,000 rpm. To remove aggregates, the cleared supernatants were loaded into microinjection syringes. Microinjection needles were pulled from thin-wall borosilicate glass capillaries. MDCK cells grown on coverslips were placed in CO2-independent medium (GIBCO-BRL) for injection. An Eppendorf Femtojet microinjection apparatus mounted on a Zeiss Axiovert 100 microscope was used to inject 200 cells per condition. In some experiments, two different cDNA were injected sequentially into the same cell nuclei by preinjecting MDCK cells with the mutant Rac1 or Cdc42 cDNA followed 5 h later by microinjection with E-cad-GFP cDNA. The sequentially injected cells were fixed and stained after 24 h when the cells had reached confluence.
Antibodies. The following primary antibodies were used: a mouse monoclonal antibody, HECD1, raised to the human E-cadherin ectodomain [provided by Alpha Yap, University of Queensland, with the permission of Dr. M. Takeichi, RIKEN (The Institute of Physical and Chemical Research), Saitama, Japan]; a rabbit polyclonal anti-E-cadherin (provided by Alpha Yap, University of Queensland); mouse monoclonal antibodies specific for p120ctn, the cis-medial Golgi-resident protein, GM130, and the Golgin family member p230 (Transduction Laboratories, Lexington, KY), which is a TGN protein; a mouse monoclonal antibody raised against lysobisphosphatidic acid (LBPA; provided by Sally Martin, University of Queensland, with permission of Jean Gruenberg, University of Geneva); and the 9E10 hybridoma cell line, which was harvested to produce monoclonal antibodies against the c-myc epitope tag. Alexa 488-conjugated phalloidin was used to stain F-actin (Molecular Probes, Eugene, OR). Cy3-conjugated sheep anti-mouse IgG secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA), and Alexa-488-conjugated goat anti-rabbit IgG secondary antibodies were purchased from Molecular Probes.
Immunofluorescence. Cells grown on coverslips were fixed in 4% paraformaldehyde, permeabilized using 0.1% Triton X-100, and incubated in a blocking buffer of 0.5% bovine serum albumin in PBS, pH 7.4. Primary and secondary antibodies were diluted in blocking buffer, and the cells were incubated for 12 h at room temperature. Coverslips were mounted onto glass microscope slides using either 90% glycerol-PBS containing 25 mg/ml 1,4-diazabicyclo[2.2.2]octane or 50% glycerol-PBS containing 1% N-propyl gallate. Cells were viewed using epifluorescence on an Olympus Provis AX-70 microscope, and images were captured using a charge-coupled device 300ET-RCX camera (DAGE-MTI, Michigan City, IN), and analyzed using Adobe PhotoShop software.
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RESULTS |
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In these experiments, overexpression of Rac1 and Cdc42 mutants by transfection accumulated some but not all of the E-cad-GFP intracellularly, possibly as a result of these GTPase mutants having only a partial or delayed effect on the trafficking of E-cad-GFP. Therefore, as an alternative approach, cells were sequentially microinjected to first express Rac1 or Cdc42 mutant cDNA, followed by injection of the E-cad-GFP cDNA at a later time. This ensured that E-cad-GFP was being made and trafficked in a preexisting environment of recombinant G protein expression (Fig. 2). In cells preexpressing RacDN, there was no surface staining of E-cad-GFP, while faint staining of the plasma membrane was still observed in cells injected with RacCA. In both cases, the majority of E-cad-GFP accumulated inside the cells was localized to large vesicular structures that were concentrated in the perinuclear region (Fig. 2A). Cdc42DN and Cdc42CA caused a near-complete absence of surface staining of E-cad-GFP in these sequential injection-expression experiments with an accumulation of E-cad-GFP inside cells (Fig. 2A). Semiquantitative image analysis of intracellular E-cad-GFP fluorescence vs. cell surface fluorescence indicated that the dominant-negative forms of Rac1 and Cdc42 had the strongest apparent effects on E-cadherin trafficking, while constitutively active Rac1 and Cdc42 were less effective in disrupting E-cad-GFP localization (Fig. 2B). Taken together, these results show that expression of Rac1 and Cdc42 mutants induced a perinuclear accumulation of E-cad-GFP and reduced cell surface staining, again consistent with an effect on E-cad-GFP trafficking.
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DISCUSSION |
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The effect of overexpressing Rac1 or Cdc42 in this study was to accumulate expressed E-cad-GFP in intracellular vesicular compartments. In previous studies, mutant forms of Cdc42 caused the intracellular accumulation of other basolateral membrane proteins (32, 38). In MDCK cells, dominant-negative forms of Cdc42, but not Rac1, selectively disrupted basolateral exocytic and endocytic trafficking, causing the mistargeting of VSVG protein (32). Furthermore, Cdc42 mutants were found to block the exit of basolateral membrane proteins such as neural cell adhesion molecule and low-density lipoprotein receptor from the TGN without affecting the polarized trafficking of soluble proteins (9, 32, 38). Our present results reveal E-cadherin as another physiologically important basolateral membrane protein whose post-Golgi transport is regulated by Cdc42. Jou and Nelson (28) observed E-cadherin in intracellular vesicles in MDCK cells expressing Rac1 mutants. In addition, a recent study (41) found that knockdown of Rac1 using small interfering RNA reduced the surface staining of E-cadherin in polarized MDCK cells, although Western blot analysis indicated that total protein levels in the cells were unchanged. Thus our data are consistent with these observations and further show that Rac1 mutants also directly affect the localization and trafficking of E-cadherin. The expression of Cdc42 mutants induces mistargeting of basolateral proteins to the apical membrane in polarized cells (9, 38). In showing that both Cdc42 and Rac1 mutants selectively disrupted the basolateral transport of E-cadherin, but not that of the missorted E-cadS1-GFP, our present findings are consistent with a specific role for Rho GTPases in basolateral trafficking (9, 38).
A possible site for Cdc42 and Rac1 to affect E-cadherin trafficking is at or near the TGN on the basis of the partial colocalization of accumulated E-cad-GFP and TGN markers. Interestingly, we consistently noted a difference in E-cad-GFP accumulated in cells expressing RacDN or Cdc42DN. Cdc42DN appears to block E-cad-GFP in a compartment overlapping more with the Golgi complex. This may imply that Cdc42 and Rac1 function in close but sequential steps to affect post-Golgi trafficking of E-cad-GFP. Setting a distal limit for the action of Rac1 and Cdc42 is the binding of p120ctn to the newly synthesized E-cadherin--catenin complex, which as we previously established happens only at or near the basolateral membrane (36). In the current experiments, p120ctn was not jointly accumulated intracellularly with E-cadherin, implying that the Rac1 block precedes the E-cadherin-p120ctn association. Our results predict that Cdc42 and Rac1 act at a subcompartment associated with or close to the TGN. The recent literature and our own data (35a) suggest that this may happen at the recycling endosome (1) or, equivalently, at the apical recycling endosome of MDCK cells (24). Ang et al. (1) reported that Cdc42 and Rab8 at the recycling endosome function in the trafficking of basolateral proteins and, moreover, that this occurs only for AP-1B-sorted basolateral proteins. The basolateral sorting of E-cadherin occurs via a dileucine-mediated, AP-1B-independent mechanism, although the specific adaptor complex involved has not been identified (36, 37). Thus a novel finding of the present study is that Cdc42 and Rac1 can regulate AP-1B-independent basolateral trafficking, implicating these Rho GTPases as regulators in more than one basolateral transport pathway. Our findings show that E-cadherin traffics through a Rab11-positive recycling endosome (35a) on its way to the cell surface and suggests this as an additional site for Cdc42 and Rac1 action.
Once at the surface, E-cadherin can be internalized and recycled to the surface or targeted to late endosomes (3). Rho GTPases also act to regulate the function and internalization of E-cadherin at the cell surface (17). In our experiments, the lack of colocalization of E-cad-GFP with LBPA in late endosomes suggests that under these conditions, Rac1 and Cdc42 accumulated E-cad-GFP in an exocytic rather than an endocytic pathway. Also, there is no evidence in these cells of the peripheral early endosome staining of E-cad-GFP typically observed during recycling of E-cadherin in MDCK cells (33). Thus the effects of Rac1 and Cdc42 shown in the present study represent functioning of these GTPases in an exocytic step in addition to their known roles at adherens junctions.
By what mechanisms do Rac and Cdc42 exert their effects on E-cadherin trafficking? Although Rho proteins can interact indirectly with the cadherin-catenin complex at the adherens junction (4), it is more likely that Rac and Cdc42 regulate E-cadherin sorting and trafficking through interactions with other components of the vesicle trafficking machinery. Cdc42, for instance, can interact with components of the exocyst to mediate basolateral trafficking (35) and participate in the Cdc42-Par6-Par3-PKC complex for polarized trafficking (25, 26). Actin is a common effector for the Rho GTPases and is also involved in post-Golgi trafficking. Vesicle budding at the TGN involves actin and myosins (5, 45). There are biochemically distinct pools of actin associated with the Golgi complex (15) and short microfilaments demarked by isoforms of the Tm5 tropomyosin gene associated with transport vesicles budding off the TGN (23, 42). Interestingly, dynamic G-actin-rich microfilaments concentrated around the Golgi area were found to disappear in cells expressing activated Cdc42 (38). Future investigations will focus on whether and how Cdc42 and Rac1 work through the polymerization of actin to regulate steps in adaptor binding or vesicle formation at the recycling endosome, with the effect of controlling the basolateral transport and cell surface delivery of E-cadherin.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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