Am J Physiol Cell Physiol AJP: Renal Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 292: C1061-C1069, 2007. First published October 4, 2006; doi:10.1152/ajpcell.00073.2006 Free Article
0363-6143/07 $8.00
This Article
Free upon publication Free Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/3/C1061    most recent
00073.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (11)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kraemer, A.
Right arrow Articles by Ali, R. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kraemer, A.
Right arrow Articles by Ali, R. G.

EXTRACELLULAR MATRIX, CELL INTERACTIONS

Rac is a dominant regulator of cadherin-directed actin assembly that is activated by adhesive ligation independently of Tiam1

Astrid Kraemer,1 Marita Goodwin,1,2 Suzie Verma,1 Alpha S. Yap,1,2 and Radiya G. Ali1,2

1Division of Molecular Cell Biology, Institute for Molecular Bioscience, and 2School for Biomedical Science, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia

Submitted 15 February 2006 ; accepted in final form 2 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Classic cadherins function as adhesion-activated cell signaling receptors. On adhesive ligation, cadherins induce signaling cascades leading to actin cytoskeletal reorganization that is imperative for cadherin function. In particular, cadherin ligation activates actin assembly by the actin-related protein (Arp)2/3 complex, a process that critically affects the ability of cells to form and extend cadherin-based contacts. However, the signaling pathway(s) that activate Arp2/3 downstream of cadherin adhesion remain poorly understood. In this report we focused on the Rho family GTPases Rac and Cdc42, which can signal to Arp2/3. We found that homophilic engagement of E-cadherin simultaneously activates both Rac1 and Cdc42. However, by comparing the impact of dominant-negative Rac1 and Cdc42 mutants, we show that Rac1 is the dominant regulator of cadherin-directed actin assembly and homophilic contact formation. To pursue upstream elements of the Rac1 signaling pathway, we focused on the potential contribution of Tiam1 to cadherin-activated Rac signaling. We found that Tiam1 or the closely-related Tiam2/STEF1 was recruited to cell-cell contacts in an E-cadherin-dependent fashion. Moreover, a dominant-negative Tiam1 mutant perturbed cell spreading on cadherin-coated substrata. However, disruption of Tiam1 activity with dominant-negative mutants or RNA interference did not affect the ability of E-cadherin ligation to activate Rac1. We conclude that Rac1 critically influences cadherin-directed actin assembly as part of a signaling pathway independent of Tiam1.

actin cytoskeleton; Cdc42; E-cadherin

CLASSIC CADHERINS are a large family of cell surface adhesion receptors that function throughout development and postembryonic life to mediate events such as cell-cell adhesion, tissue patterning, and wound healing (40, 41). This is exemplified by E-cadherin, the principal cadherin found in epithelial tissues. E-cadherin supports epithelial morphogenesis and integrity, while its dysfunction promotes tumor progression to invasion and metastasis (3). At the cellular level, many of these morphogenetic effects of cadherin activity arise through close cooperation between the adhesion receptors and the actin cytoskeleton. Thus actin cytoskeletal integrity is necessary for cadherin function (2), while several different modes of actin organization, with the potential to serve distinct cellular functions, can be found at cadherin adhesive contacts (34, 46).

One important form of cytoskeletal activity is actin assembly itself: this can potentially generate force as cells form contacts with one another and produce filamentous scaffolds for bundling and contractility (4, 19, 43). Of note, the actin-related complex (Arp)2/3 actin nucleator complex, a major determinant of actin assembly, is found at cadherin adhesions and influences the biogenesis of cell-cell contacts (19, 44). Arp2/3 can, indeed, be recruited to the cell cortex in response to adhesive ligation of the cadherin ectodomain itself. This suggests that Arp2/3 may provide local protrusive forces to drive the formation and efficient extension of cell-cell contacts (13, 19, 44).

Importantly, the catalytic activity of Arp2/3 is tightly regulated in response to cell signals (21). The signaling pathways that promote Arp2/3-mediated actin assembly in response to cadherin adhesion, however, remain poorly understood. Small GTPases of the Rho subfamily, notably Rac and Cdc42, are well-known regulators of Arp2/3 activity (21). Cdc42 was previously reported to be activated when cells form contacts with one another (6, 16), although whether this occurs in response to cadherins themselves or reflects signaling by nectin adhesion receptors remains to be determined. Rac1 localizes to newly forming cadherin adhesions, both native intercellular contacts (1, 8, 27) and homophilic adhesions that cells make to immobilized cadherin ligands (11, 18). Moreover, Rac1 is activated when cells assemble contacts with one another or adhere to cadherin-coated substrata (18, 27, 29). Inhibition of Rac1 signaling either with dominant-negative (DN) mutants or by RNA interference (RNAi)-mediated protein depletion reduced the amount of actin filaments at cell-cell contacts and prevented the formation of cadherin-based contacts (4, 30, 39). These observations therefore suggest, but do not prove, that Rac1 may be a key determinant of actin assembly at cadherin contacts. To further understand the signaling pathways that regulate the cytoskeleton at cadherin contacts, we therefore sought to directly assess the contributions of Rac1 and Cdc42 to cadherin-activated actin assembly and to identify additional molecules that may regulate signaling by these GTPases in response to cadherin adhesion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Plasmids. Plasmids used in this study were 1) a p21-activated kinase (PAK)-Cdc42 and Rac interactive binding domain (CRIB) glutathione S-transferase (GST)-fusion construct (a kind gift from Dr. J. Chernoff, Fox Chase Cancer Center, Philadelphia, PA), 2) green fluorescent protein (GFP)-tagged DN Rac1 in pCDNA3.1(+) (a kind gift from Neil Hotchin, University of Birmingham, Birmingham, UK), 3) myc-tagged DN Rac1 in pCDNA3.1(+) (a kind gift from Alan Hall, UCL, London, UK), 4) GFP-tagged DN Cdc42 in enhanced GFP (EGFP)-C1 (a kind gift from Kozo Kaibuchi, Nagoya University, Japan), 5) EGFP-tagged IRSp53 (a generous gift of Drs. Taodami Takenawa and Hiraoki Miki, Tokyo University, Japan), 6) pEGFP-N1 (Clontech), and 7) DN Tiam1 (amino acids 393–854), amplified via PCR using a 5' primer containing a EcoRV site (indicated in bold) (GAT ATC ATG AGC ACC ACC AAC AGC GAG AG) and a 3' primer including a stop (indicated by underline) and NotI site (GC GGC CGC TCA AGC TGT ATC TGA CTT CTC AAT GTG G) and ligated into pCDNA3.1(+) via EcoRV and NotI. GFP was excised from pEGFP-C1 via NheI/BglII and cloned in pCDNA3.1(+) via NheI/BamHI.

To generate a construct enabling expression of short hairpin RNA directed against Tiam1 under the control of the H1-RNA promoter a sense (GATCC CCAAG ATCGA CATGG ACGAG AAGTT CAAGA GACTT CTCGT CCATG TCGAT CTTTT TTTGG AAA) and an antisense (AGCTT TTCCA AAAAA AGATC GACAT GGACG AGAAG TCTCT TGAAC TTCTC GTCCA TGTCG ATCTT GGG) oligo were annealed and subcloned into pSUPER.retro.puro (OligoEngine) digested with BglII and HindIII. The annealed oligos contain a BglII, a HindIII, a hairpin sequence, and a termination signal (indicated in bold). As negative control a scrambled sequence was used (sense oligo GATCC CCATC GTGGA GATAT ACCTC TCATT CAAGA GATGA GAGGT ATATC TCCAC GATTT TTTGG AAA, antisense oligo AGCTT TTCCA AAAAA TCGTG GAGAT ATACC TCTCA TCTCT TGAAT GAGAG GTATA TCTCC ACGAT GGG).

Cell culture. NMuMG and NMF cells were cultured in DMEM containing 10% FCS and 10 µg/ml insulin. Generation and maintenance of Chinese hamster ovary (CHO) cells stably expressing human E-cadherin and production and purification of the recombinant protein consisting of the ectodomain of human E-cadherin fused to the Fc fragment of IgG (hE/Fc) were described previously (18, 19). Phoenix cells were maintained in DMEM supplemented with 10% FCS. CHO cells stably expressing human E-cadherin (hE-CHO) cells were transfected with Lipofectamine and PLUS reagent (Invitrogen) according to manufacturer's instructions. NMuMG cells were transfected with Lipofectamine 2000 (Invitrogen). Phoenix cells were transfected by the calcium phosphate method.

Antibodies. Primary antibodies were 1) rabbit antiserum raised against Tiam2/STEF (a kind gift from Dr. Mikio Hoshino, Kyoto University), 2) mouse MAb against Rac1 (Upstate Biotech), 3) mouse MAb against Cdc42 (BD Biosciences), 4) rabbit pAb against Tiam1 (Santa Cruz Biotechnology), 5) rabbit polyclonal antibody (PAb) against p34 (44), 6) mouse MAb against beta-tubulin (Sigma), 7) mouse MAb against E-cadherin tail region (Transduction Laboratories), 8) rabbit PAb against human E-cadherin, raised against hE/Fc (13), 9) mouse MAb against E-cadherin, clone SHE 787 (Zymed), and 10) rabbit PAb against GFP (Roche). Actin was visualized with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (Sigma). A488- and A594-labeled secondary antibodies were obtained from Molecular Probes.

Bead and planar spreading assays. Bead recruitment assays and planar spreading assays were performed as described previously (12, 18).

Immunofluorescence and microscopy. Immunofluorescence was performed as described previously (18, 19). To visualize Tiam1/Tiam2, cells were fixed with methanol for 5 min on ice. Images were acquired with a IX81 Olympus microscope equipped with an Hamamatsu Orca-ER camera. Quantification of fluorescence intensity around beads (recruitment index) was performed as previously described (44). Cell spreading was quantified in ImageJ by tracing the outline of cells with the freehand tool. The ratio of the area over the perimeter of the cell was used as indicator for cell spreading.

Rac activation assay. Expression and purification of the GST-PAK-CRIB fusion protein was performed as described previously (18). Cells were serum starved for 3 h in serum-free medium before commencement of the planar spreading assay. At various times cells were scraped off the plates, pelleted by centrifugation, and lysed in 200 µl of lysis buffer [10 mM HEPES-KOH, pH 7.4, 10 mM MgCl2, 1% Triton X-100, 10% glycerol, 1 mM Na-vanadate, and EDTA-free protease inhibitors (Roche)]. Cleared lysates were mixed with 200 µl of wash buffer (10 mM HEPES-KOH, pH 7.4, 10 mM MgCl2, 10% glycerol, 1 mM Na-vanadate, and EDTA-free protease inhibitors) and incubated with 15 µg of GST-PAK-CRIB beads at 4°C for 45 min. Beads were washed and prepared for SDS-PAGE and immunoblotting. Quantification of Rac or Cdc42 activation was performed in ImageJ with the rectangular selection tool. After background correction, the mean band intensity was normalized to the intensity at time 0 min.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
E-cadherin homophilic ligation activates Rac1 and Cdc42 signaling. As previously reported (18, 29), Rac1 is activated by cadherin homophilic ligation when cells adhere to substrata coated with recombinant cadherin ligands. To extend this analysis, we compared the response of both Cdc42 and Rac1 signaling to cadherin homophilic ligation. CHO cells stably expressing human E-cadherin (hE-CHO cells) and NMuMG cells were seeded onto plates coated with hE/Fc. At various times after attachment, GTP-loaded Rac1 and Cdc42 were precipitated from lysates by incubation with a GST fusion protein containing the CRIB domain of the Rac/Cdc42 effector PAK. In both cell lines E-cadherin ligation, but not adhesion to the nonspecific adhesive ligand poly-L-lysine (PLL), induced a transient activation of Rac1 that peaked at ~30 min (Fig. 1, A, B, and E). Both cell lines also displayed an increase in GTP-loaded Cdc42 on adhesion to hE/Fc, but not to PLL, with a time course comparable to that for Rac activation (Fig. 1, C, D, and F). Thus E-cadherin homophilic ligation was capable of activating both Rac1 and Cdc42 signaling in these experiments.


Figure 1
View larger version (52K):
[in this window]
[in a new window]

  		Fig. 1. Homophilic E-cadherin ligation activates Rac1 and Cdc42. Chinese hamster ovary (CHO) cells stably expressing human E-cadherin (hE-CHO cells; A–D) or NMuMG cells (E and F) were allowed to adhere to substrata coated with a recombinant protein consisting of the ectodomain of human E-cadherin fused to the Fc fragment of IgG (hE/Fc) or poly-L-lysine (PLL) for 0–60 min. GTP.Rac (A, B, E) or GTP.Cdc42 levels (C, D, F) were measured with p21-activated kinase (PAK)-Cdc42 and Rac interactive binding domain (CRIB) pull-down assays. In parallel, lysates were blotted for total Rac1 (A, E) or total Cdc42 (C, F). The Rac (B) or Cdc42 (D) activation responses were quantitated by expressing the level of GTP-loaded protein as a ratio of the total protein at each time point. After background correction, these ratios for GTPase activation were then normalized to the ratio at 0 min. Adhesion to hE/Fc induced a transient rise in GTP.Rac levels in both hE-CHO cells (A, B) and NMuMG cells (F). Similarly, GTP.Cdc42 levels increased transiently in both cell lines on adhesion to hE/Fc (C, D, F). In contrast, no significant changes in GTP.Rac1 or GTP.Cdc42 levels were observed when cells were plated on PLL.

 
Rac1 signaling is a dominant regulator of cadherin-activated actin assembly. Rac1 and Cdc42 are known to have profound effects on the regulation of the actin cytoskeleton, especially on Arp2/3 activity (21). Accordingly, we then turned to examination of the contribution that activated Rac1 and Cdc42 might have on actin regulation by E-cadherin. In these experiments, we used latex beads coated with hE/Fc to present spatially defined cadherin adhesive signals to cells. As reported previously (19), adhesion of hE/Fc beads, but not of concanavalin A (ConA)-coated beads, caused cellular E-cadherin to accumulate at the sites of adhesion. This was accompanied by intense accumulation of F-actin (Fig. 2, A and B), a process that requires Arp2/3 activity (13, 44). Transient expression of DN Rac1N17 (DN Rac1) reduced substantially the amount of F-actin found at cadherin contacts (Fig. 2, C and E). In contrast, DN Cdc42N17 (DN Cdc42) had only a minor effect on F-actin accumulation (Fig. 2, D and E). This suggested that, of these two potential Arp2/3-regulatory signals, Rac1 had the preponderant effect on cadherin-directed actin assembly.


Figure 2
View larger version (49K):
[in this window]
[in a new window]

  		Fig. 2. Rac is a dominant regulator of actin accumulation at sites of cadherin homophilic ligation. Cadherin-directed actin accumulation was induced by allowing beads coated with either hE/Fc (A, C, D) or concanavalin A (ConA; B) to bind to the dorsal surfaces of hE-CHO cells for 90 min. F-actin was identified by staining with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (A–D) and quantitated (E) with the recruitment index as described in MATERIALS AND METHODS. F-actin accumulation was significantly greater at sites of adhesion to hE/Fc (A, E)- compared with ConA (B)-coated beads. F-actin accumulation on stimulation of hE/Fc was substantially reduced in cells expressing myc-tagged dominant-negative (DN) Rac1 (C) but reduced to a much lesser extent in cells expressing green fluorescent protein (GFP)-tagged DN Cdc42 (D). Data are means ± SD. **P < 0.01, *P < 0.05 compared with control hE/Fc beads, 2-tailed Student's t-test.

 
When cells adhere to hE/Fc-coated substrata they spread and extend prominent cadherin-based lamellipodia (11, 18, 19, 36), a process that requires Arp2/3 activity (44). To further assess the relative contributions of Rac1 and Cdc42 to cadherin regulation of the actin cytoskeleton, we next tested their impact on cell spreading in this assay (Fig. 3). As previously described (19), phalloidin staining of control cells revealed strong accumulation of F-actin at the leading edges of lamellipodia (Fig. 3, A and B). Cells expressing DN Rac1 were able to attach to the hE/Fc substrate but failed to form lamellipodia and extend contact zones (Fig. 3, C and D). In contrast, expression of DN Cdc42 did not affect the ability of cells to spread on hE/Fc (Fig. 3, E and F). Integrin-dependent cell spreading on fibronectin, however, was inhibited by Cdc42N17 (not shown), as also observed by other investigators (31), confirming that the DN Cdc42 transgene was bioactive. Together these findings suggested that Rac1, but not Cdc42, was needed for the Arp2/3-dependent spreading of cells on cadherin-coated substrata.


Figure 3
View larger version (45K):
[in this window]
[in a new window]

  		Fig. 3. Rac but not Cdc42 signaling is required for cadherin-based cell spreading. hE-CHO cells were allowed to adhere and spread on hE/Fc-coated substrata for 90 min. Cells were transiently transfected with enhanced GFP (EGFP) alone (control) or with either GFP-tagged DN Rac1 or GFP-DN Cdc42. Cell margins were identified by TRITC-phalloidin staining (A, C, E), and spreading was quantitated by expressing cell area as a ratio of the cell perimeter (area/perimeter, G); data are means ± SD. **P < 0.01, 2-tailed Student's t-test. Control cells extended broad lamellipodia on adhesion to hE/Fc (A, B), and this was substantially reduced by expression of DN Rac1 but not by DN Cdc42 (E, F, G).

 
To then assess whether Rac1 signaling might influence Arp2/3, we tested its ability to affect Arp2/3 recruitment to cadherin homophilic adhesions. As shown in Fig. 4, A, B, and E, the p34 subunit of Arp2/3 was recruited to hE/Fc beads but showed little accumulation around control beads coated with ConA. Transient expression of DN Rac1 significantly reduced the amount of p34 recruited to hE/Fc beads (Fig. 4, C and E). In contrast, DN Cdc42 did not affect p34 recruitment (Fig. 4, D and E). This suggests that Rac1 signaling is necessary for E-cadherin adhesion to recruit Arp2/3 to the cell cortex.


Figure 4
View larger version (65K):
[in this window]
[in a new window]

  		Fig. 4. Rac signaling is required for the recruitment of Arp2/3 to cadherin homophilic adhesions. Beads coated with either hE/Fc or ConA were allowed to adhere to the dorsal surfaces of hE-CHO cells for 90 min and then fixed and stained with anti-p34 antibodies to detect the Arp2/3 complex. Prominent p34 was detected at sites of adhesion with hE/Fc-coated beads (A) but not with the ConA controls (B) and was substantially reduced by transient expression of myc-tagged DN Rac1 (C) but not DN Cdc42 (D). E: fluorescence intensity of p34 recruitment was quantified with the recruitment index as described in MATERIALS AND METHODS; data are means ± SD. **P < 0.01, 2-tailed Student's t-test compared with control hE/Fc beads.

 
Tiam1 localizes at cell-cell contacts in an E-cadherin-dependent fashion. Together these data indicated that Rac was the dominant regulator of Arp2/3 activity at cadherin contacts. We then sought to pursue regulatory proteins that might specifically determine the activation of Rac signaling by E-cadherin adhesion. Several classes of proteins control expression of Rho family GTPase signaling, notably GTPase activating proteins, guanine-nucleotide dissociation inhibitors, and guanine nucleotide exchange factors (GEFs). Of these, GEFs transform the inactive, GDP-bound conformation of a GTPase into the active, GTP-bound conformation and are the best-understood activators of signaling (33). Although there are more than 80 known or predicted Rho family GEFs (32), only two Rac1-specific GEFs, Vav2 and Tiam1, have been implicated in cadherin signaling to date (10, 14, 22).

We chose to focus on Tiam1, because it has been identified at cell-cell contacts (20) and implicated in epithelial organization (14). Tiam1 was readily identifiable by immunoblotting in NMuMG cell lysates; hE-CHO cells did not express Tiam1 but did express Tiam2/STEF (Fig. 5A), the close relative of Tiam1, which appears to share a degree of functional redundancy with Tiam1 (23, 24, 28). Moreover, endogenous Tiam1 (Fig. 5B) or Tiam2 (Fig. 5C) was clearly localized at intercellular contacts in NMuMG and hE-CHO cells, respectively. However, NMF cells, a cadherin-deficient subclone of NMuMG cells, showed only diffuse cytoplasmic localization of Tiam1 (Fig. 5B), and cadherin-negative parental CHO cells showed a similarly cytoplasmic localization of Tiam2 (Fig. 5C). Furthermore, inhibition of cadherin function in hE-CHO cells with a blocking antibody delocalized Tiam2 from intercellular contacts (Fig. 5D). Thus both Tiam1 and Tiam2 appeared to localize to cell-cell contacts in an E-cadherin-dependent fashion.


Figure 5
View larger version (109K):
[in this window]
[in a new window]

  		Fig. 5. Tiam1 and Tiam2 localize at cell-cell contacts in an E-cadherin-dependent manner. A: expression of Tiam1 or Tiam2 assessed by immunoblotting in parental CHO, hE-CHO, NMF, and NMuMG cells. Parental CHO and NMF cells do not express E-cadherin, whereas hE-CHO and NMuMG cells are cadherin positive. B: localization of endogenous Tiam1 in NMuMG and NMF cells. Clear colocalization of Tiam1 and E-cadherin was observed in NMuMG cells. In contrast, NMF cells displayed diffuse cytoplasmic staining for Tiam1 with none detected at cell-cell contacts identified by phase-contrast microscopy (arrows). C: localization of endogenous Tiam2 in parental CHO and hE-CHO cells. Tiam2 was found with E-cadherin at cell-cell contacts in hE-CHO cells but not at contacts (identified by phase-contrast microscopy) in the parental CHO cell line (arrows). D: E-cadherin-dependent localization of Tiam2 at intercellular contacts. hE-CHO cells were incubated with anti-E-cadherin antibody SHE-787 for 40 min before fixation. Under these conditions a significant amount of E-cadherin was still retained at cell junctions. However, Tiam2 localization to E-cadherin-based contacts was completely abrogated.

 
As a first test of the potential contribution of Tiam1 to cadherin signaling, we investigated whether a DN mutant could affect the ability of hE-CHO cells to spread on hE/Fc-coated substrata, our earlier data having shown that this process was highly sensitive to Rac signaling (Fig. 3; Ref. 18). We used a DN Tiam1 construct comprising the region responsible for membrane localization (PHn-CC-Ex); this construct, as well as the homologous region from Tiam2, has commonly been used to disrupt Tiam1/Tiam2 signaling (15, 23, 24, 26, 37). We found that transient expression of this mutant dramatically reduced the ability of cells to spread on hE/Fc-coated substrata (Fig. 6). In contrast, cadherin-based spreading was not altered in control cells expressing GFP alone (Fig. 6). This indicated that DN Tiam1 could readily perturb the Rac-dependent spreading of cells on cadherin substrata.


Figure 6
View larger version (42K):
[in this window]
[in a new window]

  		Fig. 6. DN Tiam1 inhibits cell spreading on cadherin substrata. Cells were allowed to adhere to hE/Fc-coated substrata to assess their ability to extend contact zones in response to E-cadherin homophilic ligation. Cell outlines were defined by TRITC-phalloidin staining and contact extension was measured with area-to-perimeter ratios (E) as described in MATERIALS AND METHODS. Cells transiently transfected with EGFP alone were able to form broad zones of adhesion (A, B). In contrast, expression of DN Tiam1 (C, D) significantly reduced contact zone extension (E; data are means ± SD). **P < 0.01, 2-tailed Student's t-test.

 
Tiam1 and Tiam2 are not essential for E-cadherin to activate Rac1. We then tested the effect of DN Tiam1 on cadherin-activated Rac1 signaling. hE-CHO cells or NMuMG cells were transiently transfected with GFP-DN Tiam1 or with GFP alone; typically we obtained >70% transfection efficiency in these experiments. Cells were then allowed to adhere to hE/Fc-coated substrata, and GTP.Rac1 levels were measured with PAK-CRIB pull-down assays. To our surprise, DN Tiam1 had no effect on Rac1 activation in either cell line (Fig. 7, A–C), despite its potent impact on cadherin-based lamellipodial extension. To confirm that the DN Tiam1 mutant could, indeed, perturb Tiam1 signaling, we analyzed the response of Rac to lysophosphatidic acid (LPA) stimulation in NMuMG cells. The LPA1 receptor, a well-known Rho activator, also activates Rac via a pathway involving Gi, phosphatidylinositol 3-kinase (PI3-kinase), and Tiam1 (42). Consistent with this, LPA treatment of NMuMG cells induced a transient rise in GTP.Rac1 levels (Fig. 7D) and expression of DN Tiam1 significantly reduced the response of GTP.Rac1 to LPA compared with GFP alone (Fig. 7E).


Figure 7
View larger version (47K):
[in this window]
[in a new window]

  		Fig. 7. E-cadherin-activated Rac signaling is unaffected by DN Tiam1. A and B: hE-CHO cells were transiently transfected with either GFP alone or GFP-tagged DN Tiam1 and then allowed to adhere to hE/Fc-coated substrata for various times before being processed for PAK-CRIB pull-down assays. Samples were separated by SDS-PAGE and immunoprobed for Rac1. Rac activation was quantitated as described in MATERIALS AND METHODS and Fig. 1. C: Rac activation was measured in NMuMG cells transfected with GFP alone or with GFP-DN Tiam1 after adhesion to hE/Fc-coated substrata. D and E: DN Tiam1 inhibits lysophosphatidic acid (LPA)-induced Rac activation. LPA (5 µM) induced transient activation of Rac1 in serum-starved NMuMG cells (D). Expression of GFP-DN Tiam1, but not GFP alone, inhibited Rac1 activation by LPA (E). All immunoblots are representative of at least 2 independent experiments.

 
To further test the role of Tiam1 in cadherin-activated Rac signaling, we then used RNAi to deplete Tiam1 levels in NMuMG cells. We restricted our efforts to NMuMG cells because the sequence of hamster Tiam2 is not yet known. Using a retroviral delivery system with high efficiency, combined with drug selection for the expressing population, we were able to reduce Tiam1 expression by >90% compared with control cells (Fig. 8A). Rac activation on binding to hE/Fc-coated substrata was measured with PAK-CRIB pull-down assays. Both Tiam1 knockdown cells [NMuMG(T–)] and control cells [NMuMG(T+)] showed a comparable transient rise in GTP.Rac with a maximum at 30 min (Fig. 8, B and C). Similarly, Tiam1 depletion did not affect the ability of cells to activate Rac1 when they reassembled contacts with one another after depletion of extracellular calcium (Fig. 8D). In contrast, the GTP.Rac response to LPA was significantly reduced in NMuMG(T–) cells compared with NMuMG(T+) cells (Fig. 8E). This, together with the fact that DN Tiam1 does not inhibit GTP-loading of Rac, indicates that Tiam1 and Tiam2 were not critical for E-cadherin ligation to activate Rac.


Figure 8
View larger version (43K):
[in this window]
[in a new window]

  		Fig. 8. Rac activation in response to E-cadherin ligation is not affected by Tiam1 depletion by RNA interference (RNAi). A: Tiam1 RNAi successfully reduces Tiam1 expression in NMuMG cells. Immunoblot of whole cell lysates from Tiam1 knockdown cells [NMuMG(T–)] and control cells [NMuMG(T+)] with a polyclonal anti-Tiam1 antibody. B and C: PAK-CRIB pull-down assays from NMuMG(T+) and NMuMG(T–) cells adhering to hE/Fc-coated substrata. D: Tiam1 RNAi does not affect Rac1 activation as cells assemble cell-cell contacts. Cell-cell contacts in monolayers of NMuMG(T+) and NMuMG(T–) cells were disrupted by chelating extracellular calcium. Rac1 activation was then measured with PAK-CRIB pull-down assays at 0–60 min after extracellular calcium was replaced to allow cells to reform contacts. E: Tiam1 RNAi successfully abolishes LPA-mediated Rac activation. PAK-CRIB pull-down assays from NMuMG(T+) and NMuMG(T–) cells exposed to LPA (5 µM, 2 min) are shown.

 
DN Tiam1 perturbs the localization of IRSp53. To gain some insight into the possible mechanism responsible for the dramatic effect of DN Tiam1 on cadherin-based cell spreading, we examined its impact on IRSp53 (Fig. 9). This Cdc42 and Rac effector is implicated in the regulation of Arp2/3 activity (38) and can associate with Tiam1 (7). As we recently reported (34), EGFP-tagged IRSp53 localizes to the leading edges of cadherin-based lamellipodia, characteristically being found continuously decorating the outer margins (Fig. 9A). In contrast, EGFP-IRSp53 was less apparent, being more discontinuously distributed at the residual lamellipodial margins of cells expressing DN Tiam1 (Fig. 9B).


Figure 9
View larger version (92K):
[in this window]
[in a new window]

  		Fig. 9. DN Tiam1 mutant perturbs localization of IRSp53 at cadherin-based lamellipodia. hE-CHO cells were transiently cotransfected with EGFP-IRSp53 and either DN Tiam1 or its vector alone. The cells were then allowed to adhere to hE/Fc-coated substrata, and the localization of EGFP-IRSp53 was identified by fluorescence microscopy. A and B: cells transfected with vector alone showed prominent localization of EGFP-IRSp53 in continuous bands at the outer margins of cadherin-based lamellipodia. C and D: in cells coexpressing DN Tiam1 (not shown) EGFP-IRSp53 was discontinuous and less prominent. B and D represent detailed views of the areas enclosed by the boxes in A and C, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
It is increasingly apparent that classic cadherins can act as adhesion-activated cell-surface receptors, capable of signaling through multiple downstream intracellular pathways (45). We have been exploring the concept that cadherins activate membrane-local signaling pathways that regulate cooperation between adhesion receptors and the actin cytoskeleton. This cooperation may involve multiple modes of actin cytoskeletal activity mediated by distinct actin regulatory proteins and signaling pathways (34). One important task is to characterize the signals that regulate actin assembly at cadherin contacts. A key element of this process involves the Arp2/3 actin nucleator that is recruited to cadherin adhesions and contributes to the biogenesis of those contacts (19, 44). Both Rac1 and Cdc42 possess a well-documented capacity to activate Arp2/3 in response to cell surface receptor signaling (21). Our present data indicate that while E-cadherin homophilic ligation can activate signaling by both these GTPases, Rac1 has the dominant impact on cadherin-activated actin assembly.

In contrast to the reasonably well-documented capacity for classic cadherins to activate Rac1, whether cadherins also signal through Cdc42 has been less clear. Cdc42 was reported to be activated when cells made contact with one another in some studies (6, 16) but not in others (10, 27). Moreover, those studies could not readily determine whether changes in GTPase signaling were immediate downstream responses to cadherin ligation or might reflect juxtacrine signaling [through, e.g., nectins (9)] that is activated when cells surfaces are brought together. Our data indicate that homophilic E-cadherin ligation, induced by allowing cells to adhere to recombinant cadherin ligands, is sufficient to activate both Cdc42 and Rac1 with similar time profiles. In contrast, neither GTPase was activated when cells adhered to nonspecific adhesive substrata. These observations thus demonstrate for the first time that E-cadherin homophilic ligation has the capacity to activate both Rac1 and Cdc42 simultaneously.

However, although both of these GTPases can activate the Arp2/3 complex, we found that Rac1 had the dominant impact on cadherin-directed actin assembly. Thus inhibition of Rac1 clearly reduced the accumulation of F-actin at sites of adhesion to cadherin-coated beads, a process that depends on Arp2/3 activity. In contrast, the minor effect of DN Cdc42 on F-actin accumulation was much less than the effect of DN Rac1. Supporting this, DN Rac1, but not DN Cdc42, profoundly inhibited the ability of cells to extend lamellipodia on cadherin-coated substrata, a functional response to homophilic ligation that requires Arp2/3 (44). Finally, expression of DN Rac1, but not DN Cdc42, inhibited the ability of E-cadherin homophilic adhesion to recruit the Arp2/3 complex itself to the cell cortex. This implies that Rac1 is the dominant GTPase signal that couples E-cadherin homophilic ligation to Arp2/3 activity. As both GTPases signal indirectly to Arp2/3, Rac-specific intermediary proteins (such as cortactin and/or WAVE) may participate in signaling from E-cadherin to the Arp2/3 complex. The precise role that Cdc42 plays in cadherin function, however, remains to be fully elucidated.

The signaling pathway that links E-cadherin ligation to Rac activation must also involve exchange factors that catalyze GTP-loading of Rac. We focused our attention on Tiam1, which has been implicated in the regulation of cadherin-based cell-cell interactions. Indeed, Tiam1 was an attractive candidate to participate in cadherin-activated Rac signaling for several reasons. First, we found that, as in earlier reports, Tiam1 (14), and additionally Tiam2, localized to cadherin-based cell-cell contacts. Moreover, our data suggest that cadherin adhesion is necessary for this localization process, as Tiam1 or -2 was lost from cell-cell contacts on treatment with function-blocking anti-cadherin antibodies, as well as in cadherin-null cells. This is consistent with earlier evidence that VE-cadherin is necessary for junctional localization of Tiam1 in endothelial cells (20). Second, Tiam1 is a PI3-kinase-responsive GEF, and cadherin-activated Rac signaling depends at least partially on PI3-kinase signaling (18, 27). Finally, we found that a DN Tiam1 mutant potently inhibited lamellipodial extension on cadherin-coated substrata, a process that requires Rac signaling.

It was therefore surprising that our subsequent studies failed to identify any role for Tiam1 in cadherin-activated Rac signaling. Neither expression of mutant Tiam1 nor depletion of Tiam1 by RNAi altered the acute rise in GTP.Rac levels that occurred when cells adhered to cadherin-coated substrata, nor did Tiam1 knockdown affect the Rac activation that occurred when cells reassembled native cell-cell contacts after depletion of extracellular calcium. Taken together, these data indicate that, although Tiam1 may be recruited to cadherin adhesions, it does not appear to be necessary for homophilic ligation to activate Rac1 signaling. Instead, it is possible that Tiam1 is recruited to cadherin contacts to regulate other aspects of junctional biogenesis, such as the assembly and integrity of tight junctions (5, 25).

How then can we account for the ability of mutant Tiam1 to inhibit cadherin lamellipodial extension? One possibility is that DN Tiam1 may bind other molecules necessary for the formation of actin-rich protrusions. Indeed, IRSp53, an adaptor protein that can link Rac and Cdc42 signaling to actin polymerization (38), was recently reported to associate with Tiam1 (7). This linkage promoted lamellipodia formation by enhancing binding of Wave2 to the complex and initiating Arp2/3 activity (7). Furthermore, when cells adhere to cadherin-coated substrata, IRSp53 localizes with Arp2/3 at the very outer margins where lamellipodia undergo extension (34). Indeed, we found that DN Tiam1 perturbed the localization of EGFP-IRSp53 at the leading edges of cadherin-based lamellipodia. This observation suggests that IRSp53 may be involved in cadherin-based spreading and that overexpression of DN Tiam1 disrupts this signaling pathway.

We conclude that Rac1 is a dominant regulator of Arp2/3-mediated actin assembly in cadherin homophilic adhesions. It is increasingly apparent that the functional impact of Arp2/3 is determined by the signals that control its precise spatial and temporal pattern of activation in cells (33). Insofar as GEFs can critically influence where and when GTPases are activated within cells, identifying the GEF that allows E-cadherin homophilic ligation to activate Rac1 remains a priority.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Queensland Cancer Fund. A. Kraemer was the recipient of a fellowship from the Deutsche Forschungsgemeinschaft, while A. S. Yap is a Senior Research Fellow of the National Health and Medical Research Council of Australia and a Research Affiliate of the Australian Research Council Special Research Center for Functional and Applied Genomics, which provided infrastructure support for this work.


   ACKNOWLEDGMENTS
 
We thank Gideon Bollag for the cDNA of human Tiam1, Taodami Takenawa and Hiroaki Miki for EGFP-IRSp53, Kris Vleminckx for NMF cells, and Mikio Hoshino for the antisera against Tiam2. As always, our colleagues in the lab provided invaluable support in so many ways.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. S. Yap, Division of Molecular Cell Biology, Institute for Molecular Bioscience, Univ. of Queensland, St. Lucia, Brisbane, Queensland, Australia 4072 (e-mail: a.yap{at}imb.uq.edu.au)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Akhtar N, Hudson KR, Hotchin NA. Co-localization of Rac1 and E-cadherin in human epidermal keratinocytes. Cell Adhes Commun 7: 465–476, 2000.[Web of Science][Medline]

2. Angres B, Barth A, Nelson WJ. Mechanism for transition from initial to stable cell-cell adhesion: kinetic analysis of E-cadherin-mediated adhesion using a quantitative adhesion assay. J Cell Biol 134: 549–557, 1996.[Abstract/Free Full Text]

3. Birchmeier W, Behrens J. Cadherin expression in carcinomas: role in the formation of cell junctions and the prevention of invasiveness. Biochim Biophys Acta 1198: 11–26, 1994.[Medline]

4. Braga VM, Machesky LM, Hall A, Hotchin NA. The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts. J Cell Biol 137: 1421–1431, 1997.[Abstract/Free Full Text]

5. Chen X, Macara IG. Par-3 controls tight junction assembly through the Rac exchange factor Tiam1. Nat Cell Biol 7: 262–269, 2005.[CrossRef][Web of Science][Medline]

6. Chu YS, Thomas WA, Eder O, Pincet F, Perez E, Thiery JP, Dufour S. Force measurements in E-cadherin-mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42. J Cell Biol 167: 1183–1194, 2004.[Abstract/Free Full Text]

7. Connolly BA, Rice J, Feig LA, Buchsbaum RJ. Tiam1-IRSp53 complex formation directs specificity of rac-mediated actin cytoskeleton regulation. Mol Cell Biol 25: 4602–4614, 2005.[Abstract/Free Full Text]

8. Ehrlich JS, Hansen MD, Nelson WJ. Spatio-temporal regulation of Rac1 localization and lamellipodia dynamics during epithelial cell-cell adhesion. Dev Cell 3: 259–270, 2002.[CrossRef][Web of Science][Medline]

9. Fukuyama T, Ogita H, Kawakatsu T, Fukuhara T, Yamada T, Sato T, Shimizu K, Nakamura T, Matsuda M, Takai Y. Involvement of the c-Src-Crk-C3G-Rap1 signaling in the nectin-induced activation of Cdc42 and formation of adherens junctions. J Biol Chem 280: 815–825, 2005.[Abstract/Free Full Text]

10. Fukuyama T, Ogita H, Kawakatsu T, Inagaki M, Takai Y. Activation of Rac by cadherin through the c-Src-Rap1-phosphatidylinositol 3-kinase-Vav2 pathway. Oncogene 25: 8–19, 2005.

11. Gavard J, Lambert M, Grosheva I, Marthiens V, Irinopoulou T, Riou JF, Bershadsky A, Mege RM. Lamellipodium extension and cadherin adhesion: two cell responses to cadherin activation relying on distinct signalling pathways. J Cell Sci 117: 257–270, 2004.[Abstract/Free Full Text]

12. Goodwin M, Kovacs EM, Thoreson MA, Reynolds AB, Yap AS. Minimal mutation of the cytoplasmic tail inhibits the ability of E-cadherin to activate Rac but not phosphatidylinositol 3-kinase: direct evidence of a role for cadherin-activated Rac signaling in adhesion and contact formation. J Biol Chem 278: 20533–20539, 2003.[Abstract/Free Full Text]

13. Helwani FM, Kovacs EM, Paterson AD, Verma S, Ali RG, Fanning AS, Weed SA, Yap AS. Cortactin is necessary for E-cadherin-mediated contact formation and actin reorganization. J Cell Biol 164: 899–910, 2004.[Abstract/Free Full Text]

14. Hordijk PL, ten Klooster JP, van der Kammen RA, Michiels F, Oomen LC, Collard JG. Inhibition of invasion of epithelial cells by Tiam1-Rac signaling. Science 278: 1464–1466, 1997.[Abstract/Free Full Text]

15. Kawauchi T, Chihama K, Nabeshima Y, Hoshino M. The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration. EMBO J 22: 4190–4201, 2003.[CrossRef][Web of Science][Medline]

16. Kim SH, Li Z, Sacks DB. E-cadherin-mediated cell-cell attachment activates Cdc42. J Biol Chem 275: 36999–37005, 2000.[Abstract/Free Full Text]

18. Kovacs EM, Ali RG, McCormack AJ, Yap AS. E-cadherin homophilic ligation directly signals through Rac and phosphatidylinositol 3-kinase to regulate adhesive contacts. J Biol Chem 277: 6708–6718, 2002.[Abstract/Free Full Text]

19. Kovacs EM, Goodwin M, Ali RG, Paterson AD, Yap AS. Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts. Curr Biol 12: 379–382, 2002.[CrossRef][Web of Science][Medline]

20. Lampugnani MG, Zanetti A, Breviario F, Balconi G, Orsenigo F, Corada M, Spagnuolo R, Betson M, Braga V, Dejana E. VE-cadherin regulates endothelial actin activating Rac and increasing membrane association of Tiam. Mol Biol Cell 13: 1175–1189, 2002.[Abstract/Free Full Text]

21. Machesky LM, Insall RH. Signaling to actin dynamics. J Cell Biol 146: 267–272, 1999.[Abstract/Free Full Text]

22. Malliri A, van Es S, Huveneers S, Collard JG. The Rac exchange factor Tiam1 is required for the establishment and maintenance of cadherin-based adhesions. J Biol Chem 279: 30092–30098, 2004.[Abstract/Free Full Text]

23. Matsuo N, Hoshino M, Yoshizawa M, Nabeshima Y. Characterization of STEF, a guanine nucleotide exchange factor for Rac1, required for neurite growth. J Biol Chem 277: 2860–2868, 2002.[Abstract/Free Full Text]

24. Matsuo N, Terao M, Nabeshima Y, Hoshino M. Roles of STEF/Tiam1, guanine nucleotide exchange factors for Rac1, in regulation of growth cone morphology. Mol Cell Neurosci 24: 69–81, 2003.[CrossRef][Web of Science][Medline]

25. Mertens AE, Rygiel TP, Olivo C, van der Kammen R, Collard JG. The Rac activator Tiam1 controls tight junction biogenesis in keratinocytes through binding to and activation of the Par polarity complex. J Cell Biol 170: 1029–1037, 2005.[Abstract/Free Full Text]

26. Michiels F, Stam JC, Hordijk PL, van der Kammen RA, Ruuls-Van Stalle L, Feltkamp CA, Collard JG. Regulated membrane localization of Tiam1, mediated by the NH2-terminal pleckstrin homology domain, is required for Rac-dependent membrane ruffling and C-Jun NH2-terminal kinase activation. J Cell Biol 137: 387–398, 1997.[Abstract/Free Full Text]

27. Nakagawa M, Fukata M, Yamaga M, Itoh N, Kaibuchi K. Recruitment and activation of Rac1 by the formation of E-cadherin-mediated cell-cell adhesion sites. J Cell Sci 114: 1829–1838, 2001.[Abstract]

28. Nishimura T, Yamaguchi T, Kato K, Yoshizawa M, Nabeshima Y, Ohno S, Hoshino M, Kaibuchi K. PAR-6-PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiam1. Nat Cell Biol 7: 270–277, 2005.[CrossRef][Web of Science][Medline]

29. Noren NK, Niessen CM, Gumbiner BM, Burridge K. Cadherin engagement regulates Rho family GTPases. J Biol Chem 276: 33305–33308, 2001.[Abstract/Free Full Text]

30. Noritake J, Fukata M, Sato K, Nakagawa M, Watanabe T, Izumi N, Wang S, Fukata Y, Kaibuchi K. Positive role of IQGAP1, an effector of Rac1, in actin-meshwork formation at sites of cell-cell contact. Mol Biol Cell 15: 1065–1076, 2004.[Abstract/Free Full Text]

31. Price LS, Leng J, Schwartz MA, Bokoch GM. Activation of Rac and Cdc42 by integrins mediates cell spreading. Mol Biol Cell 9: 1863–1871, 1998.[Abstract/Free Full Text]

32. Rossman KL, Der CJ, Sondek J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol 6: 167–180, 2005.[CrossRef][Web of Science][Medline]

33. Schmidt A, Hall A. Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. Genes Dev 16: 1587–1609, 2002.[Free Full Text]

34. Scott JA, Shewan AM, den Elzen NR, Louriero JJ, Gertler FB, Yap AS. Ena/VASP proteins can regulate distinct modes of actin organization at cadherin-adhesive contacts. Mol Biol Cell 17: 1085–1095, 2006.[Abstract/Free Full Text]

36. Shewan AM, Maddugoda M, Kraemer A, Stehbens SJ, Verma S, Kovacs EM, Yap AS. Myosin 2 is a key Rho kinase target necessary for the local concentration of E-cadherin at cell-cell contacts. Mol Biol Cell 16: 4531–4542, 2005.[Abstract/Free Full Text]

37. Stam JC, Sander EE, Michiels F, van Leeuwen FN, Kain HE, van der Kammen RA, Collard JG. Targeting of Tiam1 to the plasma membrane requires the cooperative function of the N-terminal pleckstrin homology domain and an adjacent protein interaction domain. J Biol Chem 272: 28447–28454, 1997.[Abstract/Free Full Text]

38. Suetsugu S, Kurisu S, Oikawa T, Yamazaki D, Oda A, Takenawa T. Optimization of WAVE2 complex-induced actin polymerization by membrane-bound IRSp53, PIP3, and Rac. J Cell Biol 173: 571–585, 2006.[Abstract/Free Full Text]

39. Takaishi K, Sasaki T, Kotani H, Nishioka H, Takai Y. Regulation of cell-cell adhesion by rac and rho small G proteins in MDCK cells. J Cell Biol 139: 1047–1059, 1997.[Abstract/Free Full Text]

40. Takeichi M. Morphogenetic roles of classic cadherins. Curr Opin Cell Biol 7: 619–627, 1995.[CrossRef][Web of Science][Medline]

41. Tepass U, Truong K, Godt D, Ikura M, Peifer M. Cadherins in embryonic and neural morphogenesis. Nat Rev Mol Cell Biol 1: 91–100, 2000.[CrossRef][Web of Science][Medline]

42. Van Leeuwen FN, Olivo C, Grivell S, Giepmans BN, Collard JG, Moolenaar WH. Rac activation by lysophosphatidic acid LPA1 receptors through the guanine nucleotide exchange factor Tiam1. J Biol Chem 278: 400–406, 2003.[Abstract/Free Full Text]

43. Vasioukhin V, Bauer C, Yin M, Fuchs E. Directed actin polymerization is the driving force for epithelial cell-cell adhesion. Cell 100: 209–219, 2000.[CrossRef][Web of Science][Medline]

44. Verma S, Shewan AM, Scott JA, Helwani FM, den Elzen NR, Miki H, Takenawa T, Yap AS. Arp2/3 activity is necessary for efficient formation of E-cadherin adhesive contacts. J Biol Chem 279: 34062–34070, 2004.[Abstract/Free Full Text]

45. Yap AS, Kovacs EM. Direct cadherin-activated cell signaling: a view from the plasma membrane. J Cell Biol 160: 11–16, 2003.[Abstract/Free Full Text]

46. Zhang J, Betson M, Erasmus J, Zeikos K, Bailly M, Cramer LP, Braga VM. Actin at cell-cell junctions is composed of two dynamic and functional populations. J Cell Sci 118: 5549–5562, 2005.[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 Hum Mol GenetHome page
J. Deplazes, M. Fuchs, S. Rauser, H. Genth, E. Lengyel, R. Busch, and B. Luber
Rac1 and Rho contribute to the migratory and invasive phenotype associated with somatic E-cadherin mutation
Hum. Mol. Genet., October 1, 2009; 18(19): 3632 - 3644.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. Ren, F. M. Helwani, S. Verma, R. W. McLachlan, S. A. Weed, and A. S. Yap
Cortactin Is a Functional Target of E-cadherin-activated Src Family Kinases in MCF7 Epithelial Monolayers
J. Biol. Chem., July 10, 2009; 284(28): 18913 - 18922.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
M. Hammerschmidt and D. Wedlich
Regulated adhesion as a driving force of gastrulation movements
Development, November 15, 2008; 135(22): 3625 - 3641.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
L. P. Desai, K. E. Chapman, and C. M. Waters
Mechanical stretch decreases migration of alveolar epithelial cells through mechanisms involving Rac1 and Tiam1
Am J Physiol Lung Cell Mol Physiol, November 1, 2008; 295(5): L958 - L965.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
L. Carramusa, C. Ballestrem, Y. Zilberman, and A. D. Bershadsky
Mammalian diaphanous-related formin Dia1 controls the organization of E-cadherin-mediated cell-cell junctions
J. Cell Sci., November 1, 2007; 120(21): 3870 - 3882.
[Abstract] [Full Text] [PDF]

This Article
Free upon publication Free Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/3/C1061    most recent
00073.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (11)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kraemer, A.
Right arrow Articles by Ali, R. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kraemer, A.
Right arrow Articles by Ali, R. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.