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Address correspondence to Masatoshi Takeichi, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan. Tel.: (81) 78-306-3116. Fax: (81) 78-306-3118. email: takeichi{at}cdb.riken.go.jp
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Abstract |
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Key Words: Fat; cadherin; actin cytoskeleton; Ena/VASP; cellcell interaction
Abbreviations used in this paper: EC, extracellular cadherin; EVH1, Ena/VASP homology 1; Mena, mammalian Enabled; RNAi, RNA interference; VASP, vasodilator-stimulated phosphoprotein.
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Introduction |
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Vertebrate Fat cadherin is one such molecule. Fat is the largest among the members of the cadherin superfamily, commonly having 34 EC domains. The primary sequences of the cytoplasmic regions of Fat subtypes have almost no similarity to those of the classic cadherins. In Drosophila, fat was shown to be a gene responsible for hyperplastic overgrowth of imaginal discs (Bryant et al., 1988; Mahoney et al., 1991), and recent analyses revealed that Drosophila Fat regulates planar polarity patterning (Rawls et al., 2002; Strutt and Strutt, 2002; Yang et al., 2002; Fanto et al., 2003; Ma et al., 2003). In mammals, three subtypes of Fat, namely Fat1, 2, and 3, have been reported (Dunne et al., 1995; Ponassi et al., 1999; Cox et al., 2000; Inoue et al., 2001; Mitsui et al., 2002; Nakayama et al., 2002). We can predict that mammalian Fat might also play roles in cell proliferation or planar cell polarity. However, the cytoplasmic region is not highly conserved between the Drosophila and mammalian Fats, although their extracellular regions are similar. Thus, we cannot rule out the possibility that mammalian Fat might have acquired distinct roles from its Drosophila counterpart. Fat is expressed in various tissues at embryonic stages, especially in proliferating epithelial tissues such as the neural tube, lung epithelium, and proliferating layers in the skin (Dunne et al., 1995; Ponassi et al., 1999; Cox et al., 2000; Inoue et al., 2001; Mitsui et al., 2002; Nakayama et al., 2002).
In this report, we describe for the first time analyses at both molecular and cellular levels on the properties of mammalian Fat1. Our detailed examinations revealed Fat1 to be localized at filopodia, lamellipodia, and cellcell contact sites. By performing RNA interference (RNAi) in PAM212 cells, we found that Fat1 was required for tight cellcell association and proper actin organization. Furthermore, we found that in a wound-healing assay, Fat1 was required to regulate cell polarity at the wound margins. As for its molecular action, we identified Ena/vasodilator-stimulated phosphoproteins (VASPs) as a possible downstream effector of Fat1. Fat1 bound to these proteins via an Ena/VASP homology 1 (EVH1) domainmediated interaction. We suggest that Fat1 regulates cellcell adhesion and other cell behavior by controlling actin polymerization through the Ena/VASP system, at least in part.
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Results |
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Cellcell associations are controlled by classic cadherins. To test their effects on Fat1 regulation, we treated PAM212 cells with blocking antibodies specific for E- and P-cadherins (Yoshida-Noro et al., 1984; Nose and Takeichi, 1986), the major classic cadherins expressed in this cell line. After a short period of incubation (30 min), ß-catenin localization became diffuse, most extensively at the peripheral portions of cell colonies. Coincidently, the junctional concentration of Fat1 also became diffuse or was lost entirely (Fig. 2). Thus, we suggest that Fat1 requires the classic cadherin system to become localized at cellcell junctions.
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To test the interaction between Fat1 and Ena/VASP at the cellular level, we overexpressed delN Fat1 in NRK-52E cells showing a clear focal adhesion distribution. In these cells, endogenous VASP disappeared from focal adhesions, where this protein normally localizes (Fig. 3 F, top panels), and was occasionally recruited to the sites where delN Fat1 was concentrated (unpublished data). Under these conditions, focal adhesions, as visualized by vinculin staining, still existed, suggesting that the effect of delN Fat1 was specific to Ena/VASP proteins (Fig. S2 B). However, the delN Fat1 mut, in which the Ena/VASP-binding sites had been mutated, did not exhibit such activity (Fig. 3 F, bottom panels). The expression levels of both delN Fat1 proteins and endogenous VASP in delN Fat1transfected and delN Fat1 muttransfected cells were comparable (unpublished data). Of the total number of transfectants, the percent showing the disappearance of VASP from focal adhesions was 88.8 ± 8.0% (mean ± SEM, from 10 independent experiments) for delN Fat1expressing cells, and 8.0 ± 4.0% (from 12 independent experiments) for delN Fat1 mutexpressing cells. Furthermore, when the Fat1 cytoplasmic tail attached to the mitochondrial targeting sequence of Listeria protein ActA was expressed in NRK-52E cells, endogenous VASP was recruited to the mitochondria (Fig. 3 G, top panels). However, the mutant form of Fat1, in which the Ena/VASP-binding sites had been mutated, did not have such activity (Fig. 3 G, bottom panels). These results strongly support the idea that Fat1 binds to Ena/VASP proteins specifically through an EVH1 domainmediated interaction. In addition, the Fat1 tail could not recruit N-WASP, WAVEs, vinculin, ß-catenin, or ZO-1, indicating the specific interaction between Fat1 and Ena/VASP proteins (unpublished data). Notably, the Fat1 tail was not able to induce ectopic actin fiber formation at the mitochondria, although the NH2 terminus of zyxin, which also binds to Ena/VASP proteins, had such activity in the above assays (unpublished data).
Fat1 was colocalized with VASP at cellcell contact and cell peripheries
Next, we used PAM212 cells to examine whether endogenous Fat1 was colocalized with Ena/VASP proteins. As shown in Fig. 4 (AC), these two molecules did colocalize at lamellipodia, filopodial tips, or cellcell contacts (see also Fig. 6 A). To investigate the relationship between the localization of Fat1 and Ena/VASP, we examined the behavior of these molecules in the presence of a low dose of cytochalasin D; for it had been reported that Ena/VASP disappears from lamellipodial edges by treatment with a low dose of cytochalasin D, whereas N-WASP remains (Bear et al., 2002). In the presence of 150 nM cytochalasin D, Mena and VASP all disappeared from the lamellipodial edges (Fig. 4 D, top panels; not depicted for Mena). However, the Fat1 distribution in these structures was not altered, and WAVE and N-WASP were also retained (Fig. 4 D, bottom panels; not depicted for N-WASP), suggesting that the localization of Fat1 might not be determined by Ena/VASP proteins.
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Fat1 is required for tight cell association and actin fiber organization
We used PAM212 cells, which express a high level of endogenous Fat1 (Fig. S1 B), to examine the effect of RNAi-mediated knockdown of Fat1 protein expression on cell behavior. After transfection with Fat1-RNAi plasmids, 95% of the cells in culture lost Fat1 protein, as judged by anti-Fat1 antibody staining. The remaining cells maintained high levels of Fat1 expression, allowing us to compare the properties of Fat1-positive and -negative cells side by side. Fat1-negative cells, in general, looked flatter than positive ones. Immunostaining for ß-catenin in low density cultures showed that, in the absence of Fat1, cellcell associations appeared to be significantly looser; i.e., the ß-catenin signals along cell junctions became discontinuous and were interrupted by nonjunctional intercellular spaces (Fig. 5 A). Actin organization was also severely disrupted; junctional actin bundles were disorganized (compare arrow and arrowheads in Fig. 5 B), and the number of actin stress fibers was diminished in the Fat1 knockdown cells. These abnormal actin organizations were also observed in fully confluent cultures (Fig. 5 C).
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Discussion |
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How does Fat1 control the actin cytoskeleton? We identified Ena/VASP proteins as partners of Fat1. Ena/VASP proteins are localized to free cell edges, focal adhesions, and cell junctions, where they are thought to regulate the polymerization of actin (Laurent et al., 1999; Bear et al., 2000, 2002; Krause et al., 2002; Renfranz and Beckerle, 2002). Fat1 colocalized with Ena/VASP at these sites, with the exception of focal adhesions. Ena/VASP proteins have been reported to interact with several molecules, including zyxin, vinculin, semaphorin, Robo, and Fyb/Slap, via EVH1 domainmediated interactions, and these interactions regulate the localization and activity of Ena/VASP proteins. We showed that Fat1 indeed bound to Ena/VASP proteins through an EVH1 domainmediated interaction, and further demonstrated that the Fat1 tail promoted stress fiber formation in MDCK cells in a manner dependent on its EVH1-binding regions. When Fat1 protein was eliminated from PAM212 cells by RNAi, VASP was no longer localized to cellcell contact sites; concomitantly, F-actin was reorganized into a punctate pattern at these sites. These findings suggest that the Fat1Ena/VASP interaction is one possible mechanism for the regulation of actin organization.
Loss of -catenin, a mediator for cadherinactin interaction, also abolishes the localization of Ena/VASP proteins at cellcell contacts (Vasioukhin et al., 2000). As Fat1 affected classic cadherin-mediated junctions, the effects of Fat1 deficiency on VASP localization and/or actin organization could have been indirectly produced through the perturbation of the classic cadherin system. However, in contrast to the case of
-catenin null mutants, in which junctional actin is eliminated (Vasioukhin et al., 2000), F-actin still accumulated at cell junctions in Fat1 knockdown cells, although its distribution pattern was altered, suggesting that the actin disorganization observed in Fat1 knockdown cells was not a simple consequence of the perturbation of the classic cadherin system. It is important to determine in future analyses how these two cadherin systems coordinate for actin organization. Our results also showed that the Fat1VASPactin colocalization occurred only at cell boundaries; nevertheless, Fat1 knockdown affected a broad rage of actin organization. There might be a cascade of actin reorganization that is triggered by cellcell contacts, and Fat1-knockdown may block an early step of this cascade, indirectly affecting later steps. On the other hand, it is equally possible that the Fat1 tail may have other unidentified domains or functions to cover the regulation of multiple classes of actin organization, and the Fat1VASP interaction is only part of the entire Fat1 system.
Our results indicated that the classic cadherin and Fat1 adhesion systems depended on one another for their full activities. They probably cooperate in cellcell adhesion at different sites of the lateral cell membranes, as the apical adherens junction is enriched in the classic cadherincatenin complex, whereas Fat1 is abundant at more basal sides of the lateral membranes. To elicit adhesive activity, Fat1 may undergo homophilic interactions via its cadherin motifs, as other members of the cadherin superfamily do. Such homophilic interactions may generate some intracellular signals to modulate cytoskeletal organization via the cytoplasmic tail. It remains to be elucidated what signals are elicited by Fat1 to control cell polarity, and how these are related to similar activities of the Drosophila homologue.
Fat1 is not ubiquitously expressed in the body, suggesting it has cell typespecific functions. For example, the level of Fat1 proteins greatly differed among epithelial lines, such as between PAM212 and MDCK cells. These observations suggest that this protein is not indispensable for the general control of cellcell adhesion, but rather plays some cell typespecific roles. Recently, the phenotypes of transgenic mice lacking Fat1 have been reported (Ciani et al., 2003). These mice exhibit perinatal lethality, most probably caused by loss of renal glomerular slit junctions and fusion of glomerular epithelial cell processes (podocytes). Some Fat1/ mice show holoprosencephaly and anophthalmia, which may be caused by defective cellcell interactions. We are hopeful that the present cell biological findings may aid in the interpretation of such embryonic phenotypes in the future.
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Materials and methods |
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RNAi
U6 promoter RNAi vector was provided by Dr. Y. Shi (Harvard Medical School, Boston, MA; Sui et al., 2002). The DNA sequences designed for construction of RNAi plasmid were as follows: 5'-GGACGACGGCCACTTCGAAGAG-3', a mouse Fat1 sequence, for Fat1 RNAi; 5'-GGGTGTTTACATTACACATCCA-3', a human Fat1 sequence, for control RNAi; and 5'-GGTCATGATGAGCGACTACGAG-3', another mouse Fat1 sequence, for confirming the specific effect of Fat1 RNAi. Transfections were performed by using the Amaxa electroporation system (Amaxa). By using this system, >95% cells showed a reduction in the level of Fat1 protein 48 h after the transfection.
Mutagenesis
The mutants used were constructed by PCR-based mutagenesis. PCR was performed with PfuTurbo® polymerase (Stratagene). E. coli was transformed with the DpnI restriction enzyme (Stratagene)treated PCR product. Positive clones were picked up, and mutagenesis was verified by sequencing.
Antibodies
Rabbit and rat anti-Fat1 antibodies were raised against the cytoplasmic portion of mouse Fat1 protein (residues 42014590). Affinity purification was performed using standard protocol. Other antibodies used were mouse or rabbit anti-Flag (Sigma-Aldrich), mouse anti-Mena (1:50; Transduction Laboratories), rabbit anti-VASP (1:250; Alexis), mouse anti-vinculin (1:250; Sigma-Aldrich), mouse anti-ß-catenin (1:100; 5H10, a gift from Dr. M.J. Wheelock, University of Nebraska, Omaha, NE), mouse anti-GM130 (1:250; Transduction Laboratories), and rabbit anti-WAVE (1:250; a gift from Dr. T. Takenawa, University of Tokyo, Tokyo, Japan). For double-staining experiments, rat or rabbit anti-Fat1 antibodies were used in appropriate combinations with the above antibodies. For example, when rabbit anti-VASP protein was used for staining, rat (not rabbit) anti-Fat1 was used. To block E- and P-cadherin, antibodies ECCD1 (1:250; Yoshida-Noro et al., 1984) and PCD-1 (1:250; Nose and Takeichi, 1986), respectively, were used. F-actin was visualized by use of Alexa® 488 or 568conjugated phalloidin (Molecular Probes, Inc.).
Cell cultures and transfection
MDCK, PAM212, and DLD1 cells were cultured in a 1:1 mixture of DME and Ham's F12 medium (Iwaki) supplemented with 10% FCS. NRK-52E cells (a rat renal epithelial cell line provided by S. Yonemura, RIKEN Center for Developmental Biology, Kobe, Japan) were cultured in DME (Nissui) containing 10% FCS. These cells were maintained in 5% CO2 at 37°C. Cells were transfected by using Effectine reagent (Qiagen) or FuGENETM reagent (Roche) according to the manufacturer's protocol. Alternatively, cells were transfected by using the Amaxa electroporation system (Amaxa). PAM212 cells and NRK-52E cells were very efficiently transfected by using this apparatus. For the establishment of MDCK cells constitutively expressing NH2-terminal truncated forms of Fat1, we used the pCA-Sig-IRES vector (see above). 400 µg/ml G418 was used for selecting transfected cells. Selected cells were maintained as a noncloned population.
Immunostaining
Cells were fixed with 3.7% formaldehyde in PBS for 15 min at RT. The fixed cells were then permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked with 3% BSA in PBS for 30 min at 37°C. Thereafter, the cells were incubated with appropriate antibodies in 3% BSA in PBS for 1.5 h at 37°C. Next, the cells were washed three times with PBS and incubated with fluorochrome-conjugated secondary antibodies (1:500, Alexa Fluor® secondary antibodies; Molecular Probes, Inc.) in 3% BSA in PBS for 1 h at 37°C. For rat anti-Fat1 antibody, a Cy3-conjugated secondary antibody (1:500; CHEMICON International) was used. In double staining for Fat1 and other antigens, either the rat or rabbit anti-Fat1 antibody was chosen, depending on the antibody type used for the other antigen. After three washes with PBS and two washes with Milli-Q water (Millipore), coverslips were mounted with FluorSaveTM reagent (Calbiochem). Preparations were analyzed using a laser-scanning confocal microscope (LSM 510 mounted on an Axiovert 100M microscope; Carl Zeiss MicroImaging, Inc.) on an inverted stand using C-Apochromat 40x/1.20 and Plan-Apochromat 63x/1.40 objectives (Carl Zeiss MicroImaging, Inc.) at RT. Images for presentation were prepared with Adobe Photoshop® software.
Western blotting
Fetal mouse brains or cells were lysed in 50 mM Hepes (pH 7.4) containing 2 mM EGTA, 2 mM MgCl2, 10% glycerol, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and 20 µg/ml aprotinin. Proteins were fractionated by SDS-PAGE using a 215% gradient gel (Daiichi Pure Chemicals; in Fig. 3 C, Fig. S1 and Fig. S2) or a 10% gel (in Fig. 3). The fractionated proteins were electroblotted onto immobilion-P polyvinyldifluoride membranes (Millipore) by using either a wet or a semi-dry transfer apparatus (Bio-Rad Laboratories). The membrane was blocked with 5% skim milk for 30 min at RT. Then, proteins were probed for 16 h at 4°C with antibody against rat anti-Fat1 antibody (1:100), rabbit anti-Fat1 antibody (1:100), or anti-Flag antibody (1:500) in 20 mM Tris-HCl containing 3% BSA and 150 mM NaCl. The membrane was then washed three times at RT (15 min each time) in 20 mM Tris-HCl containing 150 mM NaCl and 0.5% Tween 20 (TBS-Tween), and was subsequently incubated for 2 h at RT with secondary antibody in 20 mM Tris-HCl containing 5% skim milk and 150 mM NaCl. After three washes with TBS-Tween, the proteins were detected using the ECL plusTM reagent (PerkinElmer) according to the manufacturer's protocol.
GST pull-down assay
Cells were lysed in 50 mM Hepes (pH 7.4) containing 2 mM EGTA, 2 mM MgCl2, 10% glycerol, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and 20 µg/ml aprotinin. The lysates were then incubated with GST-recombinant proteins and GSH beads for 12 h. After the beads had been washed three times with lysis buffer, the bound proteins were separated by SDS-PAGE and analyzed by immunoblotting.
Wound-healing assay
PAM212 cells were plated in a high density and were cultured for 3 d. Monolayers of the cells were wounded by scraping with the tip of a plastic pipette.
Online supplemental material
Characterization of anti-Fat1 antibodies is described in detail. Fig. S1 shows characterization of antibodies raised against the cytoplasmic portion of Fat1. Fig. S2 shows that delN Fat1 displaces endogenous VASP, but not vinculin in Fig. 3 F. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200403006/DC1.
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Acknowledgments |
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This work was supported by a grant from the program Grants-in-Aid for Specifically Promoted Research of Ministry of Education, Science, Sports, and Culture of Japan to M. Takeichi, and by a grant-in-aid for scientific research from the Japan Society for Promotion of Science to T. Tanoue, who was also supported by the Special Postdoctoral Researcher program of RIKEN.
Submitted: 1 March 2004
Accepted: 15 April 2004
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