1 Division of Basic Sciences and Program in Developmental Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
2 Department of Zoology, University of Washington, Seattle, WA 98195, USA
*Author for correspondence (e-mail: susanp{at}fhcrc.org)
Accepted 24 May 2002
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SUMMARY |
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Key words: Rho GTPase, Drosophila melanogaster, Adherens junction, p120ctn, -catenin
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INTRODUCTION |
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The subcellular localization of Rho proteins has been shown to play an important role in their function (Symons and Settleman, 2000). Rho is predominantly cytosolic but translocates to the membrane upon ligand stimulation, with different ligands leading to differential localization (Fleming et al., 1996
). Membrane localization is dependent on a carboxy-terminal CAAX motif which allows the protein to be isoprenylated (Fleming et al., 1996
; Kranenburg et al., 1997
). Interfering with this translocation event affects the ability of Rho to carry out a subset of its functions. Prenylation of mammalian RhoB is required for it to transform NIH3T3 cells (Lebowitz et al., 1997
), while the RhoA CAAX domain is needed for cytoskeletal contraction in response to ligand stimulation in N1E-115 neuroblastoma cells (Kranenburg et al., 1997
). However, RhoBs ability to activate transcription from the c-fos serum response element and RhoAs stress fiber induction is not compromised in these systems, indicating that membrane localization is not necessary for all Rho functions.
In epithelial and endothelial cells, Rho is enriched at sites of actin accumulation and membrane ruffling, where it co-localizes with the ezrin/radixin/moesin (ERM) family of proteins (Takaishi et al., 1995). ERM family proteins are involved in mediating association of the plasma membrane and the actin cytoskeleton. The colocalization of Rho with ERM proteins suggests a role for both in the regulation of adhesive complexes. Consistent with this, the formation of cadherin-based adherens junctions (AJs) has been shown to be regulated by Rho and Rac in mammalian cell culture (Braga et al., 1997
).
In addition to the cadherins themselves, AJs contain a number of associated proteins including members of the catenin family: ß-catenin (Armadillo), -catenin, and p120 catenin (p120ctn) (Hatzfeld, 1999
; Steinberg and McNutt, 1999
). While ß-catenin and
-catenin are involved in linking cadherins to the actin cytoskeleton, p120ctn appears to play more of a regulatory role and has been proposed to both positively and negatively regulate adhesion, although the precise mechanisms involved are not yet known (Anastasiadis and Reynolds, 2000
; Noren et al., 2000
). In addition to their roles at adherens junctions, ß-catenin and p120ctn function in the nucleus in conjunction with transcription factors to regulate gene expression (Behrens et al., 1996
; Daniel and Reynolds, 1999
). Recently p120ctn has been suggested to negatively regulate the activity of Rho when present in the cytoplasm by acting as a GDI and preventing the exchange of GDP for GTP, although p120ctn has no sequence homology to other GDIs (Anastasiadis et al., 2000
). p120ctn could also be involved in activating Rho by recruiting it to sites of cadherin localization, where it can become accessible to GEFs and its downstream effectors.
Cadherin-based AJs are responsible for many of the cell-cell contacts found in the Drosophila embryo (Tepass et al., 1996; Uemura et al., 1996
). We have previously characterized a mutation in the Drosophila RhoA homolog, Rho1 (Magie et al., 1999
). This mutation results in a number of maternal and zygotic defects in morphogenetic processes consistent with a role for Rho1 in regulating cytoskeletal dynamics and cell shape changes, as well as in patterning events involving transcriptional activation. Here we show that Rho1 protein accumulates at AJs in the Drosophila embryo and ovary and that cadherin and catenin localization is aberrant in Rho1 mutants. We find that Rho1 interacts physically with p120ctn and
-catenin, components of adherens junction complexes. These data suggest a role for the catenins in recruiting Rho1 to the plasma membrane, and a subsequent role for Rho1 in the regulation of proper AJ formation.
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MATERIALS AND METHODS |
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Antibody production and characterization
BALB/c BYJ Rb(8.12) 5BNR/J mice (Jackson Labs) were immunized with GST-Rho1. The P1D9 monoclonal line was generated in the FHCRC Hybridoma Production Facility as described (Hoffstrom and Wayner, 1995; Wayner et al., 1989
), and has been sent to the Developmental Studies Hybridoma Bank (University of Iowa). Western blotting was used to test antibody specificity using bacterially expressed GST-dRho1, GST-RhoL (obtained by PCR and subcloned into pGEX-3X as a 5'BamHI-3'EcoRI fragment), GST-Rac1 and GST-Cdc42. Wild-type Drosophila whole cell (from 0- to 2-hour embryos) and nuclear (from 0- to 12-hour embryos) extracts were a gift from T. Tsukiyama. Polyclonal antiserum against Drosophila p120ctn (p120ctn) was generated by immunizing BALB/c BYJ Rb(8.12) 5BNR/J mice with a protein composed of GST fused to the first 222 aa (amino acids) of Drosophila p120ctn (CG17484).
Immunofluorescence
Embryos were prepared and detection of proteins was performed as described previously (Parkhurst et al., 1990). Ovaries were fixed for 10 minutes in 6% formaldehyde, 16.7 mM KPO4 (pH 6.8), 75 mM KCl, 25 mM NaCl and 3.3 mM MgCl2, then washed 3x in PBS + 0.1% Triton X-100. Ovarioles were dissected by hand, then blocked in 2% goat normal serum for 2 hours before incubation with primary antibody. Antisera used were as follows: anti-DE-cadherin from H. Oda (1:100), anti-neurexinIV from H. Bellen (1:15000), anti-
-spectrin from D. Branton (1:500), anti-ß-galactosidase from J. Scully (1:1000), anti-Lava lamp from J. Sisson (1:5000), and anti-Armadillo from M. Peifer and the Developmental Studies Hybridoma Bank (1:100). Anti-Rho1 P1D9 monoclonal antiserum was used at 1:50. Propidium iodide staining was done by incubating embryos in 1 µg/ml propidium iodide and 50 µg/ml RNase for 1 hour following antibody staining, then washing as described previously (Parkhurst et al., 1990
).
In vitro interactions
cDNAs corresponding to DE-cadherin (CG3722) (also known as shotgun; sht) -catenin (CG17947), armadillo (CG11579), p120ctn (CG17484), rok (CG9774) and RhoGDI (CG7823) were obtained from the Berkeley Drosophila Genome Project (BDGP). The DE-cadherin cytoplasmic domain was amplified by PCR, then subcloned as a 5'BamHI-3'EcoRI fragment into pGEX-3X. A full-length p120ctn clone was generated by overlapping PCR with a truncated cDNA (lacking exon 4) and exon 4 obtained by PCR from genomic DNA. The
-catenin, armadillo, and p120ctn ORFs were subcloned into pCite 4c+ (Novagen) as 5'SalI-3'NotI, 5'BamHI-3'XhoI, and 5'SalI-3'NotI fragments, respectively. rok and RhoGDI ORFs were subcloned into pCite 4a+ (Novagen) as 5'BamHI-3'NotI, and 5'BamHI-3'EcoRI fragments, respectively. They were then expressed from the T7 promoter using the Promega TnT in vitro expression kit. Full-length GST-Rho1 was described previously (Magie et al., 1999
). Pieces of Rho1 were amplified by PCR from the Rho1 cDNA (N-term=aa 1-75; C-term=aa 76-192; piece A=aa 1-27; piece C=aa 50-75). Substitutions were made within the full-length Rho1 cDNA using primers that change the codons corresponding to the appropriate amino acids (V14A; N17A; F39A; E40N; KDQ/A, substituting alanines at aa 27-29; KQVE/A, substituting alanines at aa 51-54; RLRP/A, substituting alanines at aa 68-71). The Rho1 pieces and substitutions were cloned into pGEX-3X as 5'BamHI-3'EcoRI fragments. His-p120ctn was generated by subcloning the p120ctn ORF into pRSetA (Invitrogen) as a 5'XhoI-3'KpnI fragment. GST pulldown assays were performed as described (Lu and Settleman, 1999
; Magie et al., 1999
).
Amino acid residues corresponding to the -catenin and p120ctn binding domains were modeled on the GTP- and GDP-bound RhoA crystal structure using the Quanta protein modeling program.
Immunoprecipitations
Embryo lysate was prepared by homogenizing an overnight collection of embryos in 0.5 ml L-buffer (PBS + 0.1% NP-40 + protease inhibitors), followed by sonication and centrifugation to pellet debris. Lysate was incubated with primary antibody in 0.5 ml L-buffer for 1 hour at 4°C. Protein G sepharose was then added and the reaction allowed to proceed overnight. Analysis was conducted using SDS-PAGE followed by western blots.
RNA interference (RNAi)
p120ctn, -catenin and fushi tarazu (ftz) single-stranded RNAs (ssRNA) were transcribed from the T7 and SP6 promoters present on the pOT2A plasmids (BDGP) using the RiboMAX RNA production system (Promega). RNA preparation and injection was conducted as described previously (Kennerdell and Carthew, 1998
). Embryos aged 15-45 minutes were injected at roughly 50% egg length with double-stranded RNA (dsRNA; 5 µM). The embryos were aged to the appropriate stage, then fixed and stained as described previously (Magie et al., 1999
).
Germline transformation
The UAS-p120ctn and UAS--catenin expression constructs were made by first amplifying the ORFs by PCR from the appropriate catenin cDNA, then cloning them into the pUASp vector (Rorth, 1998
) as 5'Kpn-3'Xba fragments. The UAS-p120ctn and UAS-
-catenin vectors (500 µg/ml) were injected along with the pTURBO helper plasmid (100 µg/ml) (Mullins et al., 1989
) into isogenic w1118 flies as described (Spradling, 1986
). Transgenics were scored by eye color and the insertions were mapped and balanced using standard genetic methods.
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RESULTS |
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Rho1 protein is expressed ubiquitously during development
Previous studies in cell culture have suggested that the subcellular localization of Rho can have important effects on some of its functions, particularly those involving cell-cell adhesion. We used the P1D9 monoclonal antibody to examine the localization of Rho1 during Drosophila embryogenesis. During the syncytial blastoderm stages, Rho1 subcellular localization changes in a cell-cycle dependent manner. During interphase, Rho1 surrounds nuclei asymmetrically (Fig. 2A), while in metaphase it accumulates at the transient furrows that form around each nucleus (Fig. 2B). At the cellular blastoderm stage Rho1 protein is present throughout the cytoplasm of all cells, but concentrated apically (Fig. 2C, arrow in Fig. 2C'). This pattern of apical localization is similar to that of the cell-cell adhesion molecule DE-cadherin, which is concentrated at AJs (Fig. 2D,D') (Oda et al., 1994). Rho1 is excluded from nuclei (Fig. 2E) and localizes to the cytoplasm, as indicated by the overlap in staining with the cytoplasmic protein Lava Lamp (Fig. 2F) (Sisson et al., 2000
). High levels of protein are also seen at the basal surface of cells underlying the pole cells in the posterior of the embryo (Fig. 2H). At later stages Rho1 protein is also located throughout the cytoplasm of cells, but in addition to showing a diffuse, uniform pattern, it is concentrated in occasional punctate spots (Fig. 2I-J'). Rho1 protein is enriched in the neural commissures of the central nervous system (Fig. 2K), and also accumulates at wound sites, such as those resulting from the microinjection of early embryos (Fig. 2L), implicating Rho1 in the wound healing response.
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Rho1 protein accumulates at sites of adherens junction formation in the embryo
Rho has been implicated in the regulation of cell adhesion through its effects on a number of different types of cellular junctions, including integrin-based focal adhesions (Schwartz and Shattil, 2000) and cadherin-based AJs (Kaibuchi et al., 1999
). We examined Rho1 localization relative to DE-cadherin, a component of AJs located around the apical margin of cells and Neurexin, a component of septate junctions (SJs), which are thought to be analogous to tight junctions in mammalian cells (Baumgartner et al., 1996
). Since AJs and other cell-cell contacts are not yet fully formed at the cellular blastoderm stage, we examined cells of the gut epithelium, which show a clear apical-basal polarity with AJs located at the apical end of the cell and SJs more basal (Fig. 3J) (Tepass and Hartenstein, 1993
). As in blastoderm embryos (Fig. 2), Rho1 protein is ubiquitously cytoplasmic but concentrated apically. Significantly, the apical cytoplasmic accumulation of Rho1 protein overlaps with the sites of cadherin localization (Fig. 3C). Neurexin is localized basal to the apical accumulations of Rho1 and does not show substantial overlap (Fig. 3F).
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Cadherin and catenin localization is disrupted in Rho1 mutants
To determine whether the accumulation of Rho1 protein at sites of DE-cadherin localization has a functional role, we examined DE-cadherin expression in Rho1 mutants. In wild-type embryos, DE-cadherin protein is localized to AJs in the apical cortex of epithelial cells, resulting in an antibody staining pattern that outlines cells distinctly (Fig. 4A-B,E-F). In zygotic Rho1 mutant embryos, however, this pattern of cadherin localization is disrupted (Fig. 4C-D,G-H). Rather than being restricted to cell-cell contacts, cadherin protein is diffusely distributed across the cell. This is especially evident in the cells near the leading edge of the epithelia undergoing dorsal closure (arrow in Fig. 4H), the process whereby the lateral epidermal cells zip together at the dorsal midline. The mislocalization of DE-cadherin in Rho1 mutants does not appear to be simply the result of cellular disorganization, as localization of the septate junction protein Neurexin is not disrupted (compare Fig. 4J with 4L). In addition, expression of other proteins such as ß-tubulin and those detected by -phosphotyrosine antibodies are not disrupted in Rho1 mutants (Magie et al., 1999
). We do not observe a similar disruption of DE-cadherin staining in Rho1 mutant ovaries, probably because we can only reduce, but not eliminate, Rho1 activity during oogenesis.
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Rho1 interacts directly with p120ctn and -catenin
To determine if the localization of Rho1 at AJs is due to direct interactions between Rho1 and components of the junctional complex, we examined the ability of Rho1 to bind the catenins using an in vitro GST-pulldown assay. As reported previously, we find that in vitro translated (IVT-) p120ctn and ß-catenin, but not -catenin, bind to the intracellular domain of DE-cadherin (Fig. 5A) (Aberle et al., 1994
; Daniel and Reynolds, 1995
; Herrenknecht et al., 1991
; Hulsken et al., 1994
). While Rho1 does not bind ß-catenin, we were surprised to find that it binds directly to both p120ctn and
-catenin. As would be expected for a protein reported to keep Rho in a GDP-bound state, p120ctn binds preferentially to GST-Rho1GDP. Unexpectedly, Rho1 also binds directly to
-catenin independently of the phosphorylation state of the nucleotide Rho1 is associated with, suggesting a role for
-catenin in either recruiting Rho1 to AJs, or tethering it there once recruited by p120ctn. We also examined the binding of Rho-kinase (Rok), a known Rho effector, and a putative RhoGDI identified by the Berkeley Drosophila Genome Project (BDGP) based on sequence homology, as controls. As expected, Rok bound preferentially to GST-Rho1GTP, while RhoGDI bound exclusively to GST-Rho1GDP (Fig. 5A).
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To verify that the in vitro interactions we observed occur in vivo, we co-immunoprecipitated Rho-containing complexes from Drosophila embryo lysates. The P1D9 monoclonal antibody immunoprecipitates endogenous Rho1 (data not shown). Western blot analysis of these Rho1-immunoprecipitated complexes reveals the presence of -catenin and p120ctn (Fig. 5C).
We mapped the domains of Rho1 required for interaction with the two catenins by using a series of Rho1 protein fragments followed by targeted amino acid substitution mutations in the context of full-length Rho1 (Fig. 5D). The Rho1 binding domains for the two catenins are distinct. -catenin maps to a surface-exposed region between the phosphate-binding loop and the effector domain that has not so far been assigned a function (Fig. 5E).
-catenin binding can be greatly reduced by substituting alanines for 3 amino acids (aa 27-29) within this domain (KDQ/A; Fig. 5D,F), but is not affected by other small substitutions (Fig. 5D,F). We also tested point mutations within the effector loop previously shown to affect binding of RhoA to particular effectors in mammalian cell culture (Sahai et al., 1998
), but none of these affected
-catenin binding.
p120ctn binds preferentially to a region between the effector domain and the switch II domain (Fig. 5D-F). This binding can be disrupted by the KQVE/A substitution (Fig. 5F). These residues are part of a surface-exposed loop distinct from that to which -catenin binds (Fig. 5E). This region changes conformation depending which nucleotide is bound, consistent with the preference of p120ctn for GDP-bound Rho1. Also consistent with this preference, p120ctn binds to dominant negative Rho1 (N17A) but not to constitutively active Rho1 (V14A) (Fig. 5F). As controls, we examined the binding of Rok and RhoGDI to these Rho mutants. Rok binding is disrupted by dominant negative Rho1, and RhoGDI by constitutively active Rho1, as expected given their nucleotide preference. Both bind as well to all other mutants tested as they do to wild-type Rho1 (Fig. 5F).
Rho1 localization is disrupted in catenin mutants
p120ctn has recently been proposed to negatively regulate Rho in the cytoplasm (Anastasiadis et al., 2000; Noren et al., 2000
). We find that embryos homozygous for a deficiency that uncovers p120ctn show a severe dorsal open phenotype and, unlike wild type (Fig. 2J'), an accumulation of Rho1 protein in the leading edge cells (Fig. 6A-B'; arrows in A',B'). Since this deficiency removes several genes, and specific p120ctn mutations have not yet been reported, we used dsRNA interference (RNAi) to specifically disrupt p120ctn function (Kennerdell and Carthew, 1998
). RNAi-generated p120ctn mutant embryos exhibit severe morphogenetic defects, particularly in head involution. This phenotype is more severe than homozygous deficiency embryos, probably due to removal of maternal as well as zygotic p120ctn by the RNAi method. In cases where the leading edge cells were detectable, an accumulation of Rho1 protein was observed in and around those cells (data not shown). To confirm this result, we injected p120ctn dsRNA into embryos expressing ß-galactosidase under the control of the puckered promoter, a gene expressed in the leading edge cells. Rho1 protein accumulates in and around the cells expressing ß-gal (Fig. 6C-F'). This accumulation is not seen in uninjected embryos (data not shown) or in embryos injected with a control dsRNA (ftz, Fig. 6G-H'). Accumulation of Rho1 protein at the leading edge is not a general property of dorsal closure mutants, as Rho1 accumulation is not observed in the dorsal closure mutants basket (Fig. 6I,K) and hemipterous (Fig. 6J,L).
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Overexpression of p120ctn or -catenin enhances the Rho1 phenotype
The observation that localization of the catenins is disrupted in embryos lacking Rho1 and Rho1 localization is aberrant in embryos with reduced p120ctn or -catenin suggests that the balance of Rho1 to the catenins is important for their proper function. If this is the case, then perturbing this balance by overexpressing the catenins should show effects in a Rho1 mutant background. We therefore generated transgenic flies containing p120ctn or
-catenin under the control of the Gal4-inducible UAS promoter that would allow us to ectopically express the catenins. One copy of UAS-p120ctn or UAS-
-catenin overexpressed in a wild-type background using an actin-Gal4 driver results in approximately 15% lethality, with a third of these embryos exhibiting very mild segmentation defects (data not shown). 25% of Rho1 homozygous mutant embryos show segmentation defects (20% mild, 5% severe; Fig. 7A). p120ctn and
-catenin enhance both the incidence and severity of the segmentation defects observed when overexpressed in a Rho1 mutant background, with
-catenin exerting the stronger effect (Fig. 7A). The enhancement effects of p120ctn and
-catenin are specific, as overexpression of GFP-tagged actin using this method had no effect on the severity of the Rho1 phenotype (Fig. 7A). 42% of Rho1 mutants overexpressing p120ctn (26% mild, 16% severe) and 51% of Rho1 mutants overexpressing
-catenin (17% mild, 34% severe) show segmentation defects. Significantly, 22% of embryos overexpressing
-catenin in a Rho1 mutant background show cuticular holes, whereas only 2% of Rho1 mutants and 3% of those overexpressing
-catenin in a wild-type background display this phenotype.
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DISCUSSION |
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We find that Rho1 activity is required to properly localize DE-cadherin during development, consistent with data from mammalian cell culture experiments implicating Rho and Rac in cadherin assembly and maintenance (Braga et al., 1999; Braga et al., 1997
). The defects we observe in cadherin localization are most prevalent in and around the leading edge (LE) cells undergoing dorsal closure. Previously Rho1 had been implicated in dorsal closure via its regulation of the LE actin cytoskeleton in cells flanking the segment borders (Harden et al., 1999
). However, the disruption we observe in cadherin distribution suggests that regulation of cell-cell adhesion may play a role in the dorsal closure phenotype observed in these embryos. Thus Rho1s effects on cadherin localization could be the result of a direct role in DE-cadherin clustering, or an indirect effect on the cortical actin cytoskeleton. The process of AJ formation in keratinocytes has been shown to require actin polymerization and the interdigitation of filopodia from neighboring cells (Vasioukhin et al., 2000
). A similar interdigitation of filopodia is seen during dorsal closure in Drosophila and is likely involved in forming adhesive contacts between the two epithelial fronts (Jacinto et al., 2000
). Since Rho and Cdc42 have been shown to act antagonistically in the formation of cellular processes in neurons (Kozma et al., 1997
), it is possible that disrupting the balance of Rho1 and Cdc42 function in LE cells results in inappropriate regulation of filopodial extensions. This could partially explain the disruption of DE-cadherin localization we observe in Rho1 mutants. Alternatively, Rho1s primary role could be in directly regulating the adhesion of cells near the LE, with Rac and Cdc42 acting as the major organizers of the acto-myosin network.
In addition to the accumulation of Rho1 protein at sites of cadherin localization, we observe a direct physical interaction between Rho1 and both p120ctn and -catenin. The catenin family of proteins is important in regulating cadherin-based adhesion and linking cadherins to the actin cytoskeleton (Kemler, 1993
). ß-catenin binds to the catenin-binding domain of the cadherin molecule as well as to
-catenin.
-catenin, in turn, acts as a link to the actin cytoskeleton, either by directly binding actin filaments or through association with other actin-binding proteins.
-catenin also has been shown to bind spectrin, a major component of the membrane skeleton underlying the plasma membrane involved in stabilizing it and determining cell shape. Human colon carcinoma Clone A cells that contain mutant
-catenin have defects in spectrin assembly (Pradhan et al., 2001
). Consistent with this, we observe a breakdown of the
-spectrin cytoskeleton in embryos injected with
-catenin dsRNA, especially in morphogenetically active cells early in gastrulation.
-catenin protein is enriched at adherens junctions, but is not as strictly localized to them as is DE-cadherin. Binding of
-catenin to Rho1 may be a general mechanism through which Rho1 is recruited to the plasma membrane.
p120ctn regulates the adhesive properties of cadherin complexes through its binding to the juxtamembrane domain of the cadherin molecule, although the precise mechanisms underlying this function are not known. p120ctn also acts in the cytoplasm where it has been proposed to negatively regulate Rho activation in a manner similar to the GDI proteins, which prevent Rho from exchanging GDP for GTP, although it shares no sequence homology with them (Anastasiadis et al., 2000). The binding of p120ctn to cadherins and its effects on Rho function have been shown to be mutually exclusive, such that once p120ctn binds a cadherin molecule, it is no longer capable of inhibiting Rho activity or function (Anastasiadis et al., 2000
). Rho would then be accessible to activating regulatory proteins such as GEFs, and could carry out its downstream functions. The physical interaction we observe between Rho1 and p120ctn suggests that this negative regulation of Rho1 is due to direct binding of p120ctn to GDP-Rho1. Interestingly, this is the same face of the Rho protein that has been shown to bind to classical GDIs (Hoffman et al., 2000
), consistent with the idea that despite the lack of sequence homology, p120ctn may be acting in a similar way. Overexpression of p120ctn in mammalian cells leads to an inhibition of Rho activity (Anastasiadis et al., 2000
; Noren et al., 2000
). Overexpression of p120ctn in our system enhances the Rho1 mutant phenotype, as would be expected for a negative regulator. We find that embryos homozygous for a deficiency uncovering the p120ctn locus show an accumulation of Rho1 protein at the leading edge and exhibit a severe dorsal open phenotype. A similar accumulation of Rho1 protein is observed in embryos injected with p120ctn dsRNA. A positive feedback mechanism may be functioning whereby the relief of p120ctn-mediated regulation in those cells results in the upregulation of Rho1 protein or an increase in Rho1 stability. It has recently been shown that overexpression of a RhoGDI in the hearts of mouse embryos results in the upregulation of RhoA expression, suggesting the existence of a negative feedback mechanism in the regulation of RhoA levels (Wei et al., 2002
), although we are not aware of any other instances in which a positive feedback mechanism has been linked to Rho expression. Excess Rho activity disrupts cellular migration (Nobes and Hall, 1999
); cells at the leading edge in embryos that lack p120ctn function remain cuboidal, rather than elongating as they would during normal dorsal closure, suggesting that Rho1 may be involved in regulating these cell shape changes. Alternatively, p120ctn has been suggested to activate Rac and Cdc42 in the cytoplasm through an interaction with the GEF Vav2, and this could account for some of its effects on cell morphology (Noren et al., 2000
). The observation that Rho1 can bind both p120ctn and
-catenin and that their binding sites are not overlapping suggests that either could be involved in recruiting Rho1 to AJs or the plasma membrane in general. Our data indicating that overexpression of
-catenin enhances the Rho1 mutant phenotype to a greater degree than p120ctn suggests an important role for
-catenin in Rho1 function, perhaps as a factor generally involved in localizing Rho1 to its sites of action, while p120ctn plays a more specific role at AJs.
Our data suggest a model (Fig. 7E) in which p120ctn or -catenin or both are involved in recruiting Rho1 to sites of cadherin localization, where it can then be activated and carry out its functions, including proper AJ formation. If Rho1 is not recruited properly, as in the case of a Rho1 mutant, this results in mislocalization of AJ components. The binding of p120ctn to Rho1, either in the cytoplasm or while Rho1 is tethered at AJs through its interaction with
-catenin, inhibits the exchange of GDP for GTP and keeps Rho1 in an inactive state. The binding of p120ctn to the juxtamembrane domain may release Rho1, allowing it to be activated by GEFs. GTP-Rho1 could then bind its downstream effectors and either directly regulate DE-cadherin assembly or maintenance, or indirectly affect AJ formation through its effects on the actin cytoskeleton. Rho1 localization at AJs could then be mediated either through continued association with
-catenin or through isoprenylation and insertion into the plasma membrane. Mutational analysis aimed at distinguishing between these models will provide further insight into this important feature of Rho1 function during morphogenesis.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Aberle, H., Butz, S., Stappert, J., Weissig, H., Kemler, R. and Hoschuetzky, H. (1994). Assembly of the cadherin-catenin complex in vitro with recombinant proteins. J. Cell Sci. 107, 3655-3663.
Anastasiadis, P. Z., Moon, S. Y., Thoreson, M. A., Mariner, D. J., Crawford, H. C., Zheng, Y. and Reynolds, A. B. (2000). Inhibition of RhoA by p120 catenin. Nature Cell Biol. 2, 637-644.[Medline]
Anastasiadis, P. Z. and Reynolds, A. B. (2000). The p120 catenin family: complex roles in adhesion, signaling and cancer. J. Cell Sci. 113, 1319-1334.
Baumgartner, S., Littleton, J. T., Broadie, K., Bhat, M. A., Harbecke, R., Lengyel, J. A., Chiquet-Ehrismann, R., Prokop, A. and Bellen, H. J. (1996). A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function. Cell 87, 1059-1068.[Medline]
Behrens, J., von Kries, J. P., Kuhl, M., Bruhn, L., Wedlich, D., Grosschedl, R. and Birchmeier, W. (1996). Functional interaction of beta-catenin with the transcription factor LEF-1. Nature 382, 638-642.[Medline]
Bishop, A. L. and Hall, A. (2000). Rho GTPases and their effector proteins. Biochem. J. 348, 241-255.[Medline]
Braga, V. M. M. (1999). Small GTPases and regulation of cadherin dependent cell-cell adhesion. J. Clin. Pathol.: Mol. Pathol. 52, 197-202.[Abstract]
Braga, V. M. M., DelMaschio, A., Machesky, L. and Dejann, E. (1999). Regulation of cadherin function by Rho and Rac: modulation by junction maturation and cellular context. Mol. Biol. Cell 10, 9-22.
Braga, V. M. M., Machesky, L. M., Hall, A. and Hotchin, N. A. (1997). The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts. J. Cell Biol. 137, 1421-1431.
Daniel, J. M. and Reynolds, A. B. (1995). The tyrosine kinase substrate p120cas binds directly to E-cadherin but not to the adenomatous polyposis coli protein or alpha-catenin. Mol. Cell. Biol. 15, 4819-4824.[Abstract]
Daniel, J. M. and Reynolds, A. B. (1999). The catenin p120(ctn) interacts with Kaiso, a novel BTB/POZ domain zinc finger transcription factor. Mol. Cell. Biol. 19, 3614-3623.
Fleming, I. N., Elliot, C. M. and Exton, J. H. (1996). Differential translocation of Rho family GTPases by lysophosphatidic acid, endothelin-1, and platelet-derived growth factor. J. Biol. Chem. 271, 33067-33073.
Hall, A. (1998). Rho GTPases and the actin cytoskeleton. Science 279, 509-514.
Halsell, S. R., Chu, B. I. and Kiehart, D. P. (2000). Genetic analysis demonstrates a direct link between Rho signaling and nonmuscle myosin function during Drosophila morphogenesis. Genetics 156, 469-480.
Harden, N., Ricos, M., Ong, Y. M., Chia, W. and Lim, L. (1999). Participation of small GTPases in dorsal closure of the Drosophila embryo: distinct roles for Rho subfamily proteins in epithelial morphogenesis. J. Cell Sci. 112, 273-284.
Hatzfeld, M. (1999). The armadillo family of structural proteins. Int. Rev. Cytol. 186, 179-224.[Medline]
Herrenknecht, K., Ozawa, M., Eckerskorn, C., Lottspeich, F., Lenter, M. and Kemler, R. (1991). The uvomorulin-anchorage protein alpha catenin is a vinculin homologue. Proc. Natl. Acad. Sci. USA 88, 9156-9160.[Abstract]
Hoffman, G. R., Nassar, N. and Cerione, R. A. (2000). Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI. Cell 100, 345-356.[Medline]
Hoffstrom, B. G. and Wayner, E. A. (1995). Immunohistochemical localization of extracellular matrix components and their receptors in human tissue. Meths. Enzymol. 245, 316-346.
Hulsken, J., Birchmeier, W. and Behrens, J. (1994). E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton. J. Cell Biol. 127, 2061-2069.[Abstract]
Jacinto, A., Wood, W., Balayo, T., Turmaine, M., Martinez-Arias, A. and Martin, P. (2000). Dynamic actin-based epithelial adhesion and cell matching during Drosophila dorsal closure. Curr. Biol. 10, 1420-1426.[Medline]
Kaibuchi, K., Kuroda, S., Fukata, M. and Nakagawa, M. (1999). Regulation of cadherin-mediated cell-cell adhesion by the Rho family GTPases. Curr. Opin. Cell Biol. 11, 591-596.[Medline]
Kemler, R. (1993). From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. Trends Genet. 9, 317-321.[Medline]
Kennerdell, J. R. and Carthew, R. W. (1998). Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled2 act in the Wingless pathway. Cell 95, 1017-1026.[Medline]
Kozma, R., Sarner, S., Ahmed, A. and Lim, L. (1997). Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid. Mol. Cell. Biol. 17, 1201-1211.[Abstract]
Kranenburg, O., Poland, M., Gebbink, M., Oomen, L. and Moolenaar, W. H. (1997). Dissociation of LPA-induced cytoskeletal contraction from stress fiber formation by differential localization of RhoA. J. Cell Sci. 110, 2417-2427.
Lebowitz, P. F., Du, W. and Prendergast, G. C. (1997). Prenylation of RhoB is required for its cell transforming function but not its ability to activate serum response element-dependent transcription. J. Biol. Chem. 272, 16093-16095.
Lu, Y. and Settleman, J. (1999). The Drosophila Pkn protein kinase is a Rho/Rac effector target required for dorsal closure during embryogenesis. Genes Dev. 13, 1168-1180.
Mackay, D. J. G. and Hall, A. (1998). Rho GTPases. J. Biol. Chem. 273, 20685-20688.
Magie, C. R., Meyer, M., Gorsuch, M. and Parkhurst, S. M. (1999). Mutations in the Rho1 small GTPase disrupt morphogenesis and segmentation during early Drosophila development. Development 126, 5353-5364.
Maruta, H. (1998). GTPase regulators: GAPs, GDSs and GDIs. In G Proteins, Cytoskeleton and Cancer (ed. H. Maruta, K. Kohama), pp. 151-170. Austin, Texas: R.G. Landes Company.
Mullins, M. C., Rio, D. C. and Rubin, G. M. (1989). cis-acting DNA sequence requirements for P-element transposition. Genes Dev. 3, 729-738.[Abstract]
Murphy, A. M. and Montell, D. J. (1996). Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis. J. Cell Biol. 133, 617-630.[Abstract]
Narumiya, S. (1996). The small GTPase Rho: Cellular functions and signal transduction. J. Biochem. 120, 215-228.[Abstract]
Narumiya, S., Watanabe, N. and Ishizaki, T. (1998). Rho Effectors: Structure and Function. In G Proteins, Cytoskeleton and Cancer (ed. H. Maruta and K. Kohama), pp. 225-237. Austin, Texas: R.G. Landes Company.
Niewiadomska, P., Godt, D. and Tepass, U. (1999). DE-Cadherin is required for intercellular motility during Drosophila oogenesis. J. Cell Biol. 144, 533-547.
Nobes, C. D. and Hall, A. (1999). Rho GTPases control polarity, protrusion and adhesion during cell movement. J. Cell Biol. 144, 1235-1244.
Noren, N. K., Liu, B. P., Burridge, K. and Kreft, B. (2000). p120ctn regulates the actin cytoskeleton via Rho family GTPases. J. Cell Biol. 150, 567-579.
Oda, H., Uemura, T., Harada, Y., Iwai, Y. and Takeichi, M. (1994). A Drosophila homolog of cadherin associated with Armadillo and essential for embryonic cell-cell adhesion. Dev. Biol. 165, 716-726.[Medline]
Parkhurst, S. M., Bopp, D. and Ish-Horowicz, D. (1990). X:A ratio is transduced by helix-loop-helix proteins in Drosophila. Cell 63, 1179-1191.[Medline]
Pradhan, D., Lombardo, C. R., Roe, S., Rimm, D. L. and Morrow, J. S. (2001). alpha-catenin binds directly to spectrin and facilitates spectrin-membrane assembly in vivo. J. Biol. Chem. 276, 4175-4181.
Rorth, P. (1998). Gal4 in the Drosophila female germline. Mech. Dev. 78, 113-118.[Medline]
Sahai, E., Alberts, A. S. and Treisman, R. (1998). RhoA effector mutants reveal distinct effector pathways for cytoskeletal reorganization, SRF activation and transformation. EMBO J. 17, 1350-1361.
Schwartz, M. A. and Shattil, S. J. (2000). Signaling networks linking integrins and Rho family GTPases. Trends Biochem. Sci. 25, 399-391.
Sisson, J. C., Field, C., Ventura, R., Royou, A. and Sullivan, W. (2000). Lava Lamp, a novel peripheral golgi protein, is required for Drosophila melanogaster cellularization. J. Cell Biol. 151, 905-917.
Spradling, A. C. (1986). P element-mediated transformation. In Drosophila, a Practical Approach (ed. D. B. Roberts), pp. 175-197. Oxford: IRL Press.
Steinberg, M. and McNutt, P. M. (1999). Cadherins and their connections: adhesion junctions have broader functions. Curr. Opin. Cell Biol. 11, 554-560.[Medline]
Symons, M. and Settleman, J. (2000). Rho family GTPases: more than simple switches. Trends Cell Biol. 10, 415-419.[Medline]
Takaishi, K., Sasaki, T., Kameyama, T., Tsukita, S., Tsukita, S. and Takai, Y. (1995). Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell-cell adhesion sites and cleavage furrows. Oncogene 11, 39-48.[Medline]
Tepass, U., Gruszynski-DeFeo, E., Haag, T. A., Omatyar, L., Torok, T. and Hartenstein, V. (1996). shotgun encodes Drosophila E-cadherin and is preferentially required during cell rearrangement in the neurectoderm and other morphogenetically active epithelia. Genes Dev. 10, 672-685.[Abstract]
Tepass, U. and Hartenstein, V. (1993). The development of cellular junctions in the Drosophila embryo. Dev. Biol. 161, 563-596.
Uemura, T., Oda, H., Kraut, R., Hayashi, S., Kataoka, Y. and Takeichi, M. (1996). Zygotic Drosophila E-cadherin expression is required for processes of dynamic epithelial cell rearrangement in the Drosophila embryo. Genes Dev. 10, 659-671.[Abstract]
Van Aelst, L. and DSouza-Schorey, C. (1997). Rho GTPases and signaling networks. Genes Dev. 11, 2295-2322.
Vasioukhin, V., Bauer, C., Yin, M. and Fuchs, E. (2000). Directed actin polymerization is the driving force for epithelial cell-cell adhesion. Cell 100, 209-219.[Medline]
Wayner, E. A., Garcia-Pardo, A., Humphries, M. J., McDonald, J. A. and Carter, W. G. (1989). Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin. J. Cell Biol. 109, 1321-1330.[Abstract]
Wei, L., Imanaka-Yoshida, K., Wang, L., Zhan, S., Schneider, M. D., DeMayo, F. J. and Schwartz, R. J. (2002). Inhibition of Rho family GTPases by Rho GDP dissociation inhibitor disrupts cardiac morphogenesis and inhibits cardiomyocyte proliferation. Development 129, 1705-1714.