The AF-6 Homolog Canoe Acts as a Rap1 Effector During Dorsal Closure of the Drosophila Embryo

Corresponding author: Ulrike Gaul, Laboratory of Developmental Neurogenetics, 1230 York Ave., New York, NY 10021., gaul{at}mail.rockefeller.edu (E-mail)

Communicating editor: T. SCHÜPBACH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Rap1 belongs to the highly conserved Ras subfamily of small GTPases. In Drosophila, Rap1 plays a critical role in many different morphogenetic processes, but the molecular mechanisms executing its function are unknown. Here, we demonstrate that Canoe (Cno), the Drosophila homolog of mammalian junctional protein AF-6, acts as an effector of Rap1 in vivo. Cno binds to the activated form of Rap1 in a yeast two-hybrid assay, the two molecules colocalize to the adherens junction, and they display very similar phenotypes in embryonic dorsal closure (DC), a process that relies on the elongation and migration of epithelial cell sheets. Genetic interaction experiments show that Rap1 and Cno act in the same molecular pathway during DC and that the function of both molecules in DC depends on their ability to interact. We further show that Rap1 acts upstream of Cno, but that Rap1, unlike Cno, is not involved in the stimulation of JNK pathway activity, indicating that Cno has both a Rap1-dependent and a Rap1-independent function in the DC process.

Rap1 belongs to the Ras superfamily of small GTPases, which cycle between an inactive GDP-bound and an active GTP-bound state, eliciting distinct downstream responses in the active state. Rap proteins were originally identified as antagonists of oncogenic Ras (KITAYAMA et al. 1989 ; COOK et al. 1993 ; OKADA et al. 1998 ; MOCHIZUKI et al. 1999 ), but more recent studies suggest that the function of Rap1 is largely independent of Ras (reviewed in ZWARTKRUIS and BOS 1999 ; BOS et al. 2001 ; CARON 2003 ). While Ras is mainly localized at the plasma membrane, Rap1 has been found in different membrane compartments, depending on the cell type. Further, Rap1 activation appears to be stimulated by numerous exchange factors that do not act on the prototypic Ras GTPases. Rap1 has been shown to act in a Ras-independent manner in the production of superoxide (BOKOCH et al. 1991 ; MALY et al. 1994 ), in cAMP-induced neurite outgrowth (YORK et al. 1998 ), and, most recently, in the regulation of integrin-mediated cell adhesion and AMPA receptor trafficking during synaptic plasticity (TSUKAMOTO et al. 1999 ; CARON et al. 2000 ; REEDQUIST et al. 2000 ; ARAI et al. 2001 ; ZHU et al. 2002 ; RANGARAJAN et al. 2003 ).

Perhaps the most important insights into the function of Rap1 are emerging from studies in Drosophila. Loss-of-function (lof) mutations in Drosophila Rap1 cause severe morphogenetic abnormalities during embryonic development, while cell proliferation and cell fate determination, processes that rely heavily on regulation by Ras, appear to be unaffected. Specifically, the ventral invagination and migration of mesodermal precursors in the embryo are severely impaired, as are head involution, dorsal closure, and the migration of gonadal precursors (ASHA et al. 1999 ). More recently, Rap1 has been shown to play a role in cell adhesion, specifically in the positioning of adherens junctions in proliferating epithelial cells (KNOX and BROWN 2002 ). These findings strongly suggest that Rap1 plays a largely Ras-independent role in cell migration and morphogenesis.

Little is currently known about the signaling pathways mediating the downstream effects of Rap1 in vertebrates or Drosophila. A number of molecules that were originally identified in vertebrates as Ras-interacting proteins, including B-Raf, members of the RalGEF family, and AF-6, were subsequently shown to associate with Rap1 as well (LINNEMANN et al. 1999 ; QUILLIAM et al. 1999 ; ZWARTKRUIS and BOS 1999 ; BOETTNER et al. 2000 ; BOS et al. 2001 ). However, the relevance of these interactions for Rap1 function in vivo remains largely unknown; to date, none of these molecules have been shown to act as Rap1 targets in an in vivo context.

Here we report that Canoe (Cno), the Drosophila ortholog of AF-6, acts as an effector of Rap1 during dorsal closure (DC) of the Drosophila embryo. DC is a morphogenetic process that occurs during midembryogenesis and involves the dorsalward movement of the lateral ectoderm over the amnioserosa, a transient structure that covers the dorsal aspect of the embryo, to enclose the embryo. This process relies entirely on the migration and elongation of ectodermal cells, without cell recruitment or proliferation, and is akin to the epithelial cell sheet movements that occur during wound healing (STRONACH and PERRIMON 1999 ). Among the genes identified as necessary for normal DC are proteins associated with the cytoskeleton and/or cell junctions and components of the Drosophila Jun N-terminal kinase (JNK) and Decapentaplegic (Dpp) pathways (STRONACH and PERRIMON 1999 ). cno is required for DC (JURGENS et al. 1984 ); its protein is localized to the adherens junction and feeds into the JNK pathway by an unknown mechanism (TAKAHASHI et al. 1998 ). Apart from the fact that it interacts with the ZO-1 homolog Tamou (TAKAHASHI et al. 1998 ), nothing is known about the regulation of Cno activity at the adherens junction.

We identified Cno as a protein that interacts with activated Rap1 in a yeast two-hybrid screen. To address the physiological relevance of this interaction, we undertook localization studies for the two proteins, a comparative phenotypic analysis, and genetic interaction experiments. We show that the Rap1 and cno loci interact synergistically in DC and that the physical interaction between Rap1 and Cno is required for DC. We further show that the role of Canoe in promoting JNK pathway activity is independent of Rap1 and that Canoe therefore has two separate functions in DC.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast two-hybrid experiments, transgenes, and genetics:
Drosophila Rap1^wt, Rap1^V12, and Rap1^N17 were fused to LexA (pBTM116); Cno fragments encoding RA1 (amino acids 1–153) or RA2 (amino acids 255–396), the Cno N terminus with both RA domains (amino acids 1–396), and an N-terminally deleted version of Cno lacking the first 361 amino acids were inserted into pGAD.

Rap1 interacting proteins were identified by screening a Drosophila embryonic GAD-fusion library with pLexA-D-Rap1^V12 as a bait, using previously described methods (VAN AELST 1998 ).

V12 and N17 versions of Drosophila Rap1 and Ras2 were generated by site-directed mutagenesis (QuickChange; Stratagene, La Jolla, CA). The Myc-epitope EQKLISEEDLNE was inserted between the second and third amino acid of Rap1 by PCR. The Cno N terminus was deleted using a primer that links codon 362 to a Kozak-embedded ATG. Wild-type and mutant Rap1, Ras2, and Cno cDNAs were inserted into pUAST (BRAND and PERRIMON 1993 ). Genotyping of embryos was based on the absence of markers carried by the balancers. For cuticle preparations, balancers carrying ubiGFP for immunohistochemistry and RNA in situ hybridization balancers carrying lacZ transgenes (CyO, wglacZ and TM3, DfdlacZ) were used. Germline clones of Rap1^CD5 were generated as described (ASHA et al. 1999 ). Cno^mis1,Rap1^CD5 and cno²,Rap1^CD5 chromosomes were generated by meiotic recombination; recombinant genotypes were determined using cno³ and Rap1^B3 alleles. cno², cno³, bsk², bsk³, and the ptc-, dpp-, pnr-, and hs-GAL4 drivers as well as the ubiGFP balancers were kindly provided by the Bloomington Drosophila Stock Center, Rap1^B3 and Rap1^CD5 by I. Hariharan, GFP-Rap1 by N. Brown, cno^mis1 by D. Yamamoto, UASbsk by M. Mlodzik, UASRas1 by D. Montell, and the TM3, DfdlacZ and CyO, wglacZ balancers by M. Baylies. Statistical significance of rescue experiments was assessed using the chi-square test.

Immunohistochemistry, RNA in situ hybridization, and cuticle preparation:
Rat polyclonal antibodies against Cno (amino acids 729–1171) were generated using standard procedures (Covance). In lieu of DRap1-specific antibodies, a transgene encoding a GFP-Rap1^wt fusion protein was expressed under the control of the endogenous Rap1 promoter (a gift from N. Brown); green fluorescent protein (GFP) was immunodetected with polyclonal anti-GFP antibodies (Molecular Probes, Eugene, OR). In addition, myc-tagged Rap1^V12 and Rap1^wt transgenic fly lines were analyzed using monoclonal anti-Myc antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-Armadillo (Anti-Arm; Developmental Studies Hybridoma Bank) marks adherens junctions, anti-Neurexin (anti-Nrx; gift from M. Bhat) marks the lateral membrane compartment, and TRITC-phalloidin visualizes the actin cytoskeleton. Secondary antibodies were from Jackson Labs and Molecular Probes. Embryos were fixed with heat/methanol (TEPASS 1996 ) and larval tissues as described in TAKAHASHI et al. 1998 . Confocal images were collected on a Zeiss LSM 510 laser scanning microscope. RNA in situ hybridizations were carried out as described in TAUTZ and PFEIFLE 1989 and cuticle preparations were as described in TAKAHASHI et al. 1998 .

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Rap1 binds Cno in a GTP-dependent manner and partially colocalizes with Cno in vivo:
To identify molecules through which Rap1 exerts its effects in Drosophila morphogenesis, we carried out a yeast two-hybrid (YTH) screen using constitutively active Drosophila Rap1^V12 as bait and a Drosophila embryonic cDNA library (0–24 hr) as the source of potentially interacting proteins (VAN AELST 1998 ). Among the clones, we found 37 cDNAs encoding cno. Cno is a multidomain protein that contains two predicted N-terminal Ras-binding domains (RA); FHA and DIL motifs that were initially described in microtubule- and actin-based motor proteins, respectively; and a PDZ domain, followed by an extended C-terminal tail with interspersed proline-rich patches (Fig 1A). Cno wild type as well as an N-terminal fragment that contains both RA domains (CnoN) binds strongly to activated Rap1^V12, but not to dominant negative Rap1^N17, as shown in two independent YTH reporter assays (Fig 1B), suggesting that Cno is a potential effector of Drosophila Rap1. This result is consistent with our previous finding that the vertebrate ortholog of Cno, AF-6, interacts in a similar fashion with mammalian Rap1 (BOETTNER et al. 2000 , BOETTNER et al. 2001 ) and indicates that the molecular mechanism of Rap1 function is conserved between flies and mammals. Further dissection of the interaction shows that both RA domains in Cno (RA1 and RA2) are able to bind to Rap1^V12 (Fig 1B). Cno also binds to the H-Ras homolog Ras1, but not to the R-Ras homolog Ras2 (data not shown).

View larger version (71K): In this window In a new window Download PPT slide   Figure 1. (a) Scheme of the Cno protein domain structure and of mutant proteins used in this study. For YTH interactions, pGAD vectors were constructed containing full-length Canoe (Cnowt), the N terminus harboring the two RA domains (CnoN), the individual RA domains (CnoRA1 and CnoRA2), and Canoe lacking the RA domains (CnoN). The asterisk indicates K-L substitutions in the RA binding domains. For fly in vivo expression, pUAS vectors were constructed containing full-length Cno, either as wild type (Cnowt) or with RA point mutations (CnoRA1*+RA2*), and Cno lacking the N terminus (CnoN). (b) YTH interactions between Rap1 and Cno using two independent reporter assays. The interaction-dependent activation of the LacZ (left) and HIS (right) genes in the L40 reporter strain was determined in duplicate after transformation with the indicated pLexA/pGAD plasmid combinations.

To examine the physiological relevance of the Rap1-Cno interaction, we conducted colocalization studies for the two proteins in the embryo at DC stages (see MATERIALS AND METHODS). We find that Cno colocalizes with the ß-catenin homolog Armadillo (Arm), which is an integral component of the adherens junction, but is not present in the basolateral membrane compartment as marked by Nrx (Fig 2, g–o). GFP-Rap1 fusion protein is found in vesicular structures in the cytoplasm and on the lateral membrane, including the adherens and septate junctions. Confocal sectioning reveals an apically located membrane compartment that contains both Rap1 and Cno proteins (Fig 2, a–f). These subcellular distributions were observed in both the lateral ectoderm and the amnioserosa, i.e., in both tissues participating in DC. We also found similar subcellular distributions for the two proteins in the wing disc epithelium using an Myc-Rap1 transgene (see MATERIALS AND METHODS; data not shown).

View larger version (69K): In this window In a new window Download PPT slide   Figure 2. Colocalization of Rap1, Cno, and junction markers Arm and Nrx. Embryonic lateral ectoderm and amnioserosa are shown in apical or basal single confocal sections, stage 13. GFP-Rap1 colocalizes with Cno to the adherens junction of both embryonic lateral ectoderm and amnioserosa (a–c); Rap1 protein, but not Cno, is present in the entire basolateral membrane compartment, as well as in the cytoplasm (d–f). Cno and Arm colocalize at the adherens junction (apical section, g–i). Nrx, which at this stage marks the entire lateral membrane compartment, colocalizes with Cno in apical sections (j–l), but not in basal sections (m–o). The localization of Cnowt (p and q) to the adherens junction is not affected by removal of the RA domains in CnoN, as determined by colocalization with Arm (r and s). cno transgenes are expressed in stripes in the embryonic ectoderm using ptcGAL4.

Thus, both proteins clearly colocalize at the adherens junction, lending support to the idea that they physically interact in vivo. The findings are consistent with our previous observation that vertebrate AF-6 and Rap1 partially colocalize at the plasma membrane in transfected epithelial MCF7 cells (BOETTNER et al. 2000 ).

Both Rap1 and cno are required for dorsal closure:
The first cno mutant alleles were isolated on the basis of their embryonic cuticle phenotype, which is characterized by defects in DC (JURGENS et al. 1984 ). The defects can be ordered into a phenotypic series, with head involution defects as weak, anterior holes of increasing size as intermediate, and complete "dorsal open" cuticles as the strongest phenotypes (Fig 3, b–d; Table 1). Even in the null allele cno², only 81% of the homozygous mutant embryos show a complete dorsal open phenotype, while the remaining 19% have anterior holes of varying size or even fully closed cuticles (Table 1). As indicated above, Cno protein is found at the adherens junctions of both amnioserosa and lateral ectoderm, suggesting that both epithelia require its function (Fig 2, a–c; TAKAHASHI et al. 1998 ). In contrast to cno mutants, Rap1 zygotic mutant embryos can survive into larval stages (ASHA et al. 1999 ), due to the presence of maternally provided Rap1. ASHA et al. 1999 have shown that removal of maternal Rap1 in germline clones leads to multiple and severe morphogenetic abnormalities, including defects in mesoderm invagination (50% of embryos), head involution (50%), and DC (10%); each of the defects increased in penetrance and severity when paternal/zygotic Rap1 was removed as well. Consistent with these findings, we observe a variety of cuticle defects in maternal Rap1 germline clones, ranging from ventral open (sometimes with an additional dorsal hole) to large anterior holes (Fig 3, e–f; see also Fig 4N and Fig O). The latter phenotype appears to represent a superimposition of head involution and ventral and dorsal closure defects. In many cases, however, the earlier defects in ventral closure and concomitant defects in head involution may actually obscure the requirement for Rap1 in DC. Specifically, tension in the lateral ectoderm is likely to be greatly reduced if the embryo is not closed ventrally, with the result that dorsal fusion requires a lot less stretching of ectoderm cells than is needed under wild-type conditions. When we examine maternal Rap1 germline clones during DC, we indeed find that many embryos close dorsally with little or no stretching of the dorsal ectoderm (Fig 4N), supporting the idea that defective ventral closure may reduce the cellular (and thus the genetic) requirements for DC.

View larger version (142K): In this window In a new window Download PPT slide   Figure 3. Embryonic cuticle phenotypes. (a) Wild type. (b–d) cno lof allelic series. Phenotypes range from head involution defect (b, arrowhead) and small anterior hole (c, arrow) to large hole covering almost the entire dorsal aspect of the embryo (d). (e–h) Rap1 lof and transgenic conditions. Maternal Rap1 germline clones show large anterior holes (e) or are ventral open (f, arrowhead), sometimes with a small additional dorsal hole (arrow). Rap1N17 embryos display large dorsal holes (g), similar to the strong cno lof phenotype. Rap1V12 embryos show mild anterior defects (h, arrow). (i–l) Genetic interaction between Rap1 and cno. Embryos heteroallelic for a weak and a strong cno allele (cnomis1/cno2) show, at worst, head involution defects (i, arrowhead) or small anterior holes (j, arrow). Removal of zygotic Rap1 from this background leads to strongly exacerbated phenotypes: Most embryos either have large anterior holes, sometimes with additional smaller dorsal holes (k, arrow), or are completely dorsal open (l). (m–p) Rescue of cno lof with cno (mutant) transgenes. Expression of cnowt almost completely rescues the cno lof defect (n); removal of the RA binding domains strongly reduces this rescue capacity (p, arrow indicates small anterior hole). Note that cno overexpression results in head involution defects (m and n, arrowheads). (q–s) Genetic interactions between Rap1 and cno, using Rap1 transgenes. Expression of cno provides substantial rescue of the Rap1N17 phenotype (q, arrowhead indicates head involution defect), but removal of the RA binding domains abolishes this rescue capacity (r). Rap1V12 is unable to rescue the cno lof phenotype (s). (t and u) Genetic interactions of cno and Rap1 with bsk. bsk expression rescues the cno lof phenotype (t), but not Rap1N17 (u). The genetic interaction and rescue experiments are quantified in Table 1.

View larger version (163K): In this window In a new window Download PPT slide   Figure 4. Cellular phenotypes of wild-type, Rap1, and cno mutants during DC. Lateral and dorsolateral views of ectoderm and amnioserosa of wild-type (a, e, and i), ptcGAL4; Rap1N17 (b, f, and j), pnrGAL4; Rap1N17 (c, g, and k), and cno (d, h, and l) embryos during early (stage 13; a–d and i–l) and late (stage 15; e–h) DC, as visualized by staining with anti-Cno or anti-Arm antibodies and phalloidin, and of wild-type (m) and Rap1 germline clone (GLC) embryos (n–p) in late DC, as visualized by anti-FasIII and phalloidin. In both ptc/pnrGAL4; UASRap1N17 and cno mutant embryos, DC begins normally, with accumulation of actin cytoskeleton at the LE (i–l) and stretching of the lateral ectoderm (a–d), but is followed by a severing of the connection between amnioserosa and lateral ectoderm and a relaxation of the cells in the lateral ectoderm (e–h). In Rap1 GLC embryos, actin cytoskeleton accumulates at the LE (p); in many cases, DC does occur, but without stretching of the lateral ectoderm (n, compare with m; see text); in some embryos, DC defects are observed (o).

To sidestep any such masking of the role of Rap1 in DC by earlier defects and to study its function more specifically in the context of DC, we generated a dominant negative version of Rap1, Rap1^N17. We expressed it in the tissues that participate in DC, namely the lateral ectoderm and the amnioserosa, using the UAS/GAL4 system with patched (ptc) and pannier (pnr) GAL4 as drivers (see MATERIALS AND METHODS; BRAND and PERRIMON 1993 ). ptcGAL4 promotes broad expression in the ectoderm and amnioserosa, with expression in the ectoderm resolving into two stripes per segment during DC, while pnrGal4 drives expression more specifically in the dorsalmost cells of the lateral ectoderm and in the amnioserosa. We obtained similar results with both drivers (compare Fig 4B, Fig F, and Fig J with Fig 4E, Fig G, and Fig K; see below). Expression of Rap1^N17 in the ectoderm and amnioserosa leads to a strong but variable DC defect, similar to that of cno lof (Fig 3G). The DC defect of Rap1^N17 is completely rescued by coexpression of Rap1^wt (Table 1). By contrast, the expression of dominant negative versions of Ras1 or Ras2, the Ras family members closest to Rap1, show little if any effect on DC, suggesting that the biochemically detectable interaction between Cno and Ras1 has no functional importance in DC [data not shown; HARDEN et al. 1999 report mild effects of Ras1^N17 and Ras1^Q13 on DC using hsGAL4, but we have not been able to reproduce these effects using Ras1^N17 and Ras1^V12 under ptcGAL4 control]. Taken together, our findings indicate that the DC phenotype of Rap1^N17 results from a disruption of Rap1 function rather than from promiscuous interference with another GTPase (cf. CARON et al. 2000 ; REEDQUIST et al. 2000 ).

Overactivity of Rap1 and cno causes phenotypic defects as well: Expression of a dominant active Rap1^V12 transgene leads to very mild DC defects (Fig 3H). Overexpression of cno^wt does not affect DC, but causes a gain-of-function (gof) head involution defect (Fig 3M), which was not analyzed in greater detail.

To further compare the phenotypes of Rap1 and cno, we carried out a cellular characterization of the DC defects of Rap1^N17, driven by either ptcGAL4 or pnrGAL4, and cno using several molecular markers. In both mutant situations, the leading edge (LE) cytoskeleton (an accumulation of actin, nonmuscle myosin, and phosphotyrosine-containing proteins in the dorsalmost row of ectodermal cells) is assembled largely as in wild type (Fig 4, i–l) and an initial stretching of ectodermal cells takes place (Fig 4, a–d). However, at later stages of DC, both types of mutant embryos show a detachment of the lateral ectoderm from the amnioserosa; the ectoderm retracts, with cells resuming a nonelongated shape, and the amnioserosa shrivels (Fig 4, e–h). This suggests that adhesion between the two structures is impaired or that the ectodermal cells are incapable of stretching sufficiently to maintain adhesion to the amnioserosa or, conversely, that the amnioserosa cells are incapable of changing shape appropriately to maintain adhesion to the ectoderm. Since Rap1 and Cno are coexpressed in both tissues, further experiments using specific drivers will be needed to determine whether their function is required in the ectoderm, in the amnioserosa, or in both tissues. Note that Cno is still found at the adherens junction in Rap1^N17 transgenic conditions, suggesting that Rap1 activity is not required for the localization of Cno. Overall, we observe strong phenotypic similarities between Rap1^N17 transgenic and cno lof conditions, at both the cuticular and the cellular level, lending further support to the idea that Rap1 and Cno participate in the same molecular mechanism.

Loss of zygotic Rap1 enhances a mild cno phenotype:
To test whether Rap1 and Cno act in the same process, we asked whether the Rap1 and cno loci interact genetically. We made use of the previous finding that the trans-heterozygous combination of a weak and a strong cno allele (cno^mis1/cno²), which shows only mild DC defects, provides a sensitive background for interacting loci (TAKAHASHI et al. 1998 ). In analyzing embryos with the heteroallelic combination, we find that 77% develop into larvae or have a completely closed cuticle, 22% have small anterior holes, and only 1% have a strong DC defect (see Fig 3I and Fig J; Table 1). Rap1 zygotic null embryos survive into larval stages without externally visible defects, due to the presence of maternally provided Rap1 (cf. ASHA et al. 1999 ). Removal of zygotic Rap1 from the heteroallelic cno background (Rap1^CD5, cno^mis1/Rap1^CD5, cno²) results in a strong exacerbation of phenotypic defects: Only 4% of embryos develop into larvae or have a completely closed cuticle, 37% have (large) anterior holes, and 59% are completely open dorsally (Fig 3K and Fig L; Table 1). This pronounced synergy between the Rap1 and cno loci in DC provides conclusive genetic evidence for the involvement of Rap1 in DC and argues that Rap1 and Cno act in the same pathway.

The interaction between Rap1 and Cno is required for dorsal closure:
To directly assess the biological significance of the physical interaction between Rap1 and Cno, we decided to disrupt the ability of the two proteins to bind to each other and examine how this affects their function in DC. To generate Cno mutant proteins deficient in Rap1 binding, we introduced point mutations in the RA domains of Cno (K57L and K274L), since mutations at corresponding sites in AF-6 (K32L and K265L) abolish the binding of AF-6 to vertebrate Ras^V12 in vitro. Individually, these mutations (Cno^RA1* and Cno^RA2*) lead to only a mild reduction in Rap1 binding in YTH assays (data not shown), while combining them (Cno^RA1*+RA2*) significantly reduces Rap1 binding (Fig 1B). However, if we remove the two RA domains located at the very N terminus of the protein (Cno^N), Rap1 binding is completely abolished in two independent YTH reporter assays (Fig 1B). This truncation leaves the other known functional domains of the protein intact. All four mutant Cno proteins can be expressed at high levels and still localize specifically to the adherens junction (Fig 2R and Fig S), suggesting that they are otherwise not detectably impaired in their function. The fact that the Cno^N protein localizes to the adherens junction confirms that Rap1 input is not necessary for the localization of Cno.

We first asked whether expression of the cno transgenes can rescue the cno lof phenotype. As a baseline, we established that overexpression of a wild-type cno transgene with a ptcGAL4 driver is sufficient to almost completely rescue the DC defects of cno lof embryos (Fig 3N; Table 1). We then compared the double mutant and deletion mutant proteins with the wild type. We find that the ability to rescue the cno lof defect is modestly reduced in cno^RA1*+RA2*: 75% of embryos show a closed cuticle, as opposed to 92% in embryos expressing cno^wt (P < 0.02). In contrast, it is strongly decreased in cno^N, with only 35% of embryos showing a closed cuticle (P < 10^-10). However, the rescue ability of cno^N is still considerable: 35% closed cuticle embryos compares to 4% in cno lof (P < 10^-10; Fig 3O and Fig P; Table 1). Thus, disruption of the Rap1-binding capacity of Cno results in reduction, but not elimination of the ability to restore Cno function. This partial rescue suggests that the role of Cno in DC is partially dependent on its ability to bind to Rap1, but also in part independent of it (see below).

In a second set of experiments we examined whether the function of Rap1 in DC shows a similar dependence on binding between Rap1 and Cno. We find that the DC phenotype of Rap1^N17 is substantially rescued by concurrent expression of cno^wt. By contrast, expression of cno^N is completely unable to rescue Rap1^N17 (Fig 3Q and Fig R; Table 1), indicating that binding between Rap1 and Cno is indeed required for the function of Rap1 in DC. The mechanistic interpretation of these two experiments hinges on whether expression of Rap1^N17 completely abolishes Rap1 activity: If it does, the rescue by cno^wt would have to be due to overactivity of the Rap1-independent aspect of Cno function, which, as shown above, possesses considerable ability to rescue the cno lof phenotype. However, the fact that deletion of the Rap1-binding domains, under the same expression conditions, completely abrogates Cno's ability to rescue Rap1^N17 argues against this possibility. This leaves the following explanation: Expression of Rap1^N17 reduces Rap1 protein activity to a very low level that is insufficient to support DC when Cno protein is present at wild-type levels, but does allow a partial rescue when Cno protein is overexpressed. Thus, Cno's ability to rescue Rap1^N17 depends entirely on its ability to bind residual active Rap1. This indicates that the Rap1-Cno interaction is critical for the function of Rap1 in DC.

Taken together, our genetic experiments clearly demonstrate that Rap1 and Cno act in the same pathway and that their physical interaction is required for the function of both molecules in DC. What, then, is their epistatic relationship? The YTH experiments suggest that Rap1 acts upstream of Cno. In a typical signal transduction pathway, in which one component regulates the localization and/or activity of the other, one expects that expression of an independently localized/active version of the upstream component is unable to rescue a null condition of the downstream component, while expression of an independently localized/active form of the downstream component rescues the null condition of the upstream component. In the case of Rap1 and Cno, the first leg of this experiment is unproblematic, since we have both a cno null condition and a constitutively active version of Rap1. We find that Rap1^wt and activated Rap1^V12 do not rescue cno lof, even though both are able to rescue Rap1^N17 (Fig 3S; Table 1); this is consistent with Rap1 acting upstream of Cno. The second leg of the epistasis experiment, however, is less straightforward due to the lack of suitable mutant proteins: As described above, the Rap1 null condition is phenotypically complex and difficult to interpret, leaving only the Rap1^N17 transgenic condition, which most likely does not abolish Rap1 activity completely. Second, we have no constitutively active/localized version of Cno whose activity is independent of Rap1. In fact, the rescue experiments described earlier show that removal of the ability to bind Rap1 markedly reduces the DC functionality of Cno.

Rap1 and cno differentially influence JNK pathway activity:
The fact that Cno^N partially rescues cno lof indicates that a considerable portion of Cno's activity in DC is Rap1 independent. To investigate this further, we examined the events downstream of Cno. Cno has been reported to act upstream of the JNK pathway (TAKAHASHI et al. 1998 ), which is an essential and the best-studied pathway involved in DC (STRONACH and PERRIMON 1999 ). The relationship between Drosophila Rap1 and the JNK pathway is largely unexplored. We therefore examined the influence of Rap1 and Cno on the JNK pathway by genetic interaction experiments and by assessing their effects on the expression of the secreted TGF-ß homolog dpp in the LE. dpp is under JNK control and is thought to be essential for the elongation of the adjacent lateral ectodermal cells (STRONACH and PERRIMON 1999 ). Examination of cno lof mutants does not reveal any significant alteration in the early expression of dpp in the LE, but shows a consistent reduction of late dpp expression in 50% of cno mutant embryos (n > 100; Fig 5C and Fig D). Overexpression of cno^wt has no discernible effect on dpp expression (data not shown), indicating that cno is partially required but not sufficient for dpp expression. Overexpression of DJNK basket (bsk) restores dpp expression to normal levels (Fig 5E) and results in a significant but partial rescue of the cno mutant DC defect (Fig 3T; Table 1). Together, these results indicate that Cno does indeed act upstream of the JNK pathway by permitting or stimulating signaling. In contrast, dpp expression is not altered by expression of Rap1^N17 or Rap1^V12 (Fig 5F and Fig G). Also, no obvious change in dpp expression is observed in Rap1 null embryos, which lack both maternal and zygotic Rap1 (Fig 5H). This is consistent with the previous finding that expression of puc, a second transcriptional target of the JNK pathway, is unaffected in embryos lacking maternal Rap1 (ASHA et al. 1999 ). Moreover, overexpression of bsk^wt does not rescue the DC defect of Rap1^N17 (Fig 3U; Table 1). Thus, while Cno plays a significant role in maintaining JNK activity during DC, Rap1 has no apparent effect on the JNK pathway. This finding is in line with the results of our genetic interaction experiments, which indicate that the function of Cno in DC is partially independent of its interaction with Rap1. Taken together, our data suggest that Cno has two separate functions during DC: The first is controlled by Rap1 and does not involve the JNK pathway; the second is independent of Rap1 and feeds into the JNK pathway.

View larger version (140K): In this window In a new window Download PPT slide   Figure 5. dpp expression in the LE of wild-type, Rap1, and cno mutants, as indicated by RNA in situ hybridization. The genotypes of embryos were determined by scoring the absence of lacZ-marked balancers (see MATERIALS AND METHODS). In wild type, LE cell-specific transcription of dpp begins in the extended germ band (stage 11, a) and is maintained during germ-band retraction (stage 13, b). In cno lof mutants, dpp levels are normal at stage 11 (c), but significantly reduced at stage 13 in 50% of the animals (d); this effect is rescued by overexpression of bsk (e). dpp expression appears to be normal in Rap1N17, Rap1V12, and Rap1 GLC embryos (f–h). Note that the overall dpp staining is stronger in h than in a–g.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Rap1 plays an important role in cell migration and morphogenesis in both vertebrates and invertebrates. In Drosophila, embryos lacking both zygotic and maternal Rap1 display strong defects in diverse morphological aspects of embryogenesis, such as ventral invagination, migration of mesodermal precursors, head involution, and DC. A key question is which effector pathways mediate the morphogenetic functions of Rap1. We used the YTH system to identify Drosophila Rap1-specific effector molecules from an embryonic library and retrieved several cDNAs encoding Cno. We found that both N-terminal Ras-binding domains (RA1 and RA2) possess Rap1-binding potential and that they interact only with a constitutively active Rap1 mutant, Rap1^V12, but not with a dominant negative version of Rap1, Rap1^N17, suggesting that Cno may act as an effector for Rap1.

We have provided several lines of evidence confirming this hypothesis. Rap1 and Cno partially colocalize at the adherens junction in the two tissues that are involved in DC, the amnioserosa and the lateral ectoderm, with Rap1 being present at the entire lateral membrane and also showing vesicular expression throughout the cytoplasm. Moreover, loss of function of the two molecules leads to similar phenotypes, at both the cuticular and the cellular level. To directly address the question whether Rap1 utilizes Cno as an effector during DC, we conducted a series of genetic experiments. They demonstrate that the two molecules act in the same pathway and that their physical interaction is essential for their function in DC: (1) Removal of zygotic Rap1 strongly enhances the phenotype of a weak heteroallelic cno combination; (2) removal of the RA-interaction domains and, thus, removal of the ability to bind Rap1, reduces the ability of cno transgenes to rescue the cno lof phenotype; and (3) removal of the RA-interaction domains eliminates the ability of cno to rescue Rap1^N17. Finally, our finding that activated Rap1^V12 fails to rescue the cno lof defects indicates that Rap1 acts upstream of Cno. Taken together, our YTH data, colocalization results, and genetic interaction experiments provide comprehensive evidence that Cno functions as a downstream effector of Rap1 in the DC process. To our knowledge, these findings represent the first demonstration of a protein acting as a Rap1 effector in vivo.

The events downstream of Rap1 and Cno, however, appear to be more complex. Several independent findings suggest that Cno's role in DC can be separated into Rap1-independent and Rap1-dependent functions: Removal of the RA-interaction domains does not affect the ability of the remainder of the protein to localize to the adherens junction, and the mutant protein retains the capacity to partially rescue the DC defect of a cno lof mutant. Further, Cno feeds into the JNK pathway, while Rap1 does not: dpp expression levels in the LE are significantly reduced in cno lof embryos at later stages of DC, but appear unaffected in Rap1 mutants. In addition, cno lof is partially rescued by overexpressing bsk (DJNK), whereas the Rap1^N17 defect is not. Given the multidomain structure of Cno, it is not surprising that the molecule would participate in multiple pathways. Such a bifurcation of the pathway would also explain the lack of transitivity that we observe in our rescue experiments: Rap1 lof is (partially) rescued by cno overexpression, cno lof is (partially) rescued by bsk overexpression, but Rap1 lof is not rescued by bsk overexpression. The fact that both cno^N and bsk are unable to rescue Rap1 lof demonstrates that the Rap1-independent function of Cno cannot compensate for the loss of Rap1. This leaves the reciprocal question of whether Rap1 may have a second, Cno-independent function in DC. The fact that the DC phenotype of Rap1^N17 is as severe as that of cno lof without affecting JNK pathway signaling might suggest that Rap1 has additional effectors in DC (as does the fact that the phenotype of Rap1^N17 is more severe than that of cno²; ptcGAL4 UAScno^N). However, we have no conclusive evidence to support this idea, since the additional effectors of Rap1 we identified in our YTH screen have not been investigated for their role in DC.

One obstacle in investigating the function of Rap1 is its pleiotropy. A detailed analysis of DC defects, in particular, is difficult to perform in Rap1 null embryos, due to the severe disruption of multiple aspects of embryonic development prior to DC. We therefore had to make use of the dominant negative Rap1^N17 mutant. When expressed at appropriate stages in the epithelial cells that are involved in the DC process, this transgene results in robust DC defects. However, early in vitro studies appeared to show that the Rap1^N17 mutant does not compete well with normal Rap1 for the GEF C3G (VAN DEN BERGHE et al. 1997 ), calling into question whether it can be regarded as a Rap1 dominant negative. But more recent in vivo studies by CARON et al. 2000 and REEDQUIST et al. 2000 and now our own clearly show that Rap1^N17 acts as a dominant negative mutant in Rap1 signaling. Our successful rescue of Rap1^N17 with a concomitantly expressed Rap1^wt transgene demonstrates the specificity of the mutant. Further, dominant negative versions of Drosophila Ras1 and Ras2, the counterparts of the mammalian H, K, and N-Ras and of the R-Ras proteins, respectively, do not disrupt DC when they are examined under the same conditions. This shows that the interaction between DRas1 and Cno that has been detected in vitro by us and others (MATSUO et al. 1997 ) and the genetic interaction between DRas1 and Cno that was found to influence cone cell formation in the Drosophila eye (MATSUO et al. 1997 ) have no role during DC.

Which cellular processes might Rap1 and Cno act on? Cno is a multidomain protein consisting of several known and putative protein-interaction domains, including the two RA domains and a PDZ domain, which targets proteins to specific cell membranes and assembles proteins into supramolecular signaling complexes, but no catalytic domain. Cno localizes to the adherens junction and may act by localizing and clustering signal transduction components at the junction (BUCHERT et al. 1999 ) or by modulating the mechanical resistance of the adherens junction, and thus, directly or indirectly, influence JNK signaling. Since Cno is found at the adherens junctions under Rap1 lof conditions as well as in the absence of its RA domains, Rap1 cannot be required for the initial localization of the Cno protein, suggesting that Rap1 influences the activity of Cno by changing its conformation. However, another possibility is suggested by KNOX and BROWN 2002 , who found that Rap1 function is required for evenly (re-)distributing adherens junction components in wing disc epithelial cells after mitosis. It is likely that the adherens junctions in the cells that undergo stretching in the embryonic ectoderm during DC are similarly subject to dynamic reorganization, which may in part be regulated by the Rap1/Cno complex. This idea would be consistent with our observation that in Rap1 and cno lof mutants the lateral ectoderm begins its dorsal stretching, but is then unable to complete the process. Interestingly, Rap1 in mammalian cells has been shown to be activated in cell-stretching assays (SAWADA et al. 2001 ). In this system, force initiation apparently results in the activation of the JNK kinase family member p38, suggesting the existence of a Rap1-dependent "mechanosensory" pathway. Our data fit this idea. Future studies using fluorescently tagged Rap1 and Cno proteins and live imaging will shed light on dynamic aspects of their localization and function during DC.

	FOOTNOTES

¹ These authors contributed equally to this work.
² Present address: Osaka Bioscience Institute, 6-2-4 Furuedai, Suita-shi, Osaka 565-0874, Japan.

	ACKNOWLEDGMENTS

We thank H. Bülow for help constructing the Drosophila YTH library; H. Hanafusa for his support in the initial phase of the project; and M. Bhat, N. Brown, R. Fehon, I. Hariharan, M. Mlodzik, the Bloomington Stock Center, and the Developmental Studies Hybridoma Bank for fly strains and reagents. We thank U. Unnerstall for help in the preparation of the figures and manuscript. This work was supported by a postdoctoral fellowship from the Deutsche Akademie der Naturforscher Leopoldina (B.B.), by a Charles H. Revson/Norman and Rosita Winston fellowship and National Cancer Institute grant 44356 (S.I.), by a grant from the National Institutes of Health (L.V.A.), and by Research Project grant RPG-00-237-01-CSM from the American Cancer Society (U.G.).

Manuscript received July 15, 2002; Accepted for publication April 24, 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ARAI, A., Y. NOSAKA, E. KANDA, K. YAMAMOTO, and N. MIYASAKA et al., 2001  Rap1 is activated by erythropoietin or interleukin-3 and is involved in regulation of beta1 integrin-mediated hematopoietic cell adhesion. J. Biol. Chem. 276:10453-10462.[Abstract/Free Full Text]

ASHA, H., N. D. DE RUITER, M. G. WANG, and I. K. HARIHARAN, 1999  The Rap1 GTPase functions as a regulator of morphogenesis in vivo. EMBO J. 18:605-615.[Medline]

BOETTNER, B., E. E. GOVEK, J. CROSS, and L. VAN AELST, 2000  The junctional multidomain protein AF-6 is a binding partner of the Rap1A GTPase and associates with the actin cytoskeletal regulator profilin. Proc. Natl. Acad. Sci. USA 97:9064-9069.[Abstract/Free Full Text]

BOETTNER, B., C. HERRMANN, and L. VAN AELST, 2001  Ras and Rap1 interaction with AF-6 effector target. Methods Enzymol. 332:151-168.[Medline]

BOKOCH, G. M., L. A. QUILLIAM, B. P. BOHL, A. J. JESAITIS, and M. T. QUINN, 1991  Inhibition of Rap1A binding to cytochrome b558 of NADPH oxidase by phosphorylation of Rap1A. Science 254:1794-1796.[Abstract/Free Full Text]

BOS, J. L., J. DE ROOIJ, and K. A. REEDQUIST, 2001  Rap1 signalling: adhering to new models. Nat. Rev. Mol. Cell Biol. 2:369-377.[Medline]

BRAND, A. H. and N. PERRIMON, 1993  Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401-415.[Abstract]

BUCHERT, M., S. SCHNEIDER, V. MESKENAITE, M. T. ADAMS, and E. CANAANI et al., 1999  The junction-associated protein AF-6 interacts and clusters with specific Eph receptor tyrosine kinases at specialized sites of cell-cell contact in the brain. J. Cell Biol. 144:361-371.[Abstract/Free Full Text]

CARON, E., 2003  Cellular functions of the Rap1 GTP-binding protein: a pattern emerges. J. Cell Sci. 116:435-440.[Abstract/Free Full Text]

CARON, E., A. J. SELF, and A. HALL, 2000  The GTPase Rap1 controls functional activation of macrophage integrin alphaMbeta2 by LPS and other inflammatory mediators. Curr. Biol. 10:974-978.[Medline]

COOK, S. J., B. RUBINFELD, I. ALBERT, and F. MCCORMICK, 1993  RapV12 antagonizes Ras-dependent activation of ERK1 and ERK2 by LPA and EGF in Rat-1 fibroblasts. EMBO J. 12:3475-3485.[Medline]

HARDEN, N., M. RICOS, Y. M. ONG, W. CHIA, and L. LIM, 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.[Abstract]

JÜRGENS, G., E. WIESCHAUS, C. NÜSSLEIN-VOLHARD, and H. KLUDING, 1984  Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. II. Zygotic loci on the third chromosome. Roux's Arch. Dev. Biol. 193:28-44.

KITAYAMA, H., Y. SUGIMOTO, T. MATSUZAKI, Y. IKAWA, and M. NODA, 1989  A ras-related gene with transformation suppressor activity. Cell 56:77-84.[Medline]

KNOX, A. L. and N. H. BROWN, 2002  Rap1 GTPase regulation of adherens junction positioning and cell adhesion. Science 295:1285-1288.[Abstract/Free Full Text]

LINNEMANN, T., M. GEYER, B. K. JAITNER, C. BLOCK, and H. R. KALBITZER et al., 1999  Thermodynamic and kinetic characterization of the interaction between the Ras binding domain of AF6 and members of the Ras subfamily. J. Biol. Chem. 274:13556-13562.[Abstract/Free Full Text]

MALY, F. E., L. A. QUILLIAM, O. DORSEUIL, C. J. DER, and G. M. BOKOCH, 1994  Activated or dominant inhibitory mutants of Rap1A decrease the oxidative burst of Epstein-Barr virus-transformed human B lymphocytes. J. Biol. Chem. 269:18743-18746.[Abstract/Free Full Text]

MATSUO, T., K. TAKAHASHI, S. KONDO, K. KAIBUCHI, and D. YAMAMOTO, 1997  Regulation of cone cell formation by Canoe and Ras in the developing Drosophila eye. Development 124:2671-2680.[Abstract]

MOCHIZUKI, N., Y. OHBA, E. KIYOKAWA, T. KURATA, and T. MURAKAMI et al., 1999  Activation of the ERK/MAPK pathway by an isoform of rap1GAP associated with G alpha(i). Nature 400:891-894.[Medline]

OKADA, S., M. MATSUDA, M. ANAFI, T. PAWSON, and J. E. PESSIN, 1998  Insulin regulates the dynamic balance between Ras and Rap1 signaling by coordinating the assembly states of the Grb2-SOS and CrkII-C3G complexes. EMBO J. 17:2554-2565.[Medline]

QUILLIAM, L. A., A. F. CASTRO, K. S. ROGERS-GRAHAM, C. B. MARTIN, and C. J. DER et al., 1999  M-Ras/R-Ras3, a transforming ras protein regulated by Sos1, GRF1, and p120 Ras GTPase-activating protein, interacts with the putative Ras effector AF6. J. Biol. Chem. 274:23850-23857.[Abstract/Free Full Text]

RANGARAJAN, S., J. M. ENSERINK, H. B. KUIPERIJ, J. DE ROOIJ, and L. S. PRICE et al., 2003  Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the {beta}2-adrenergic receptor. J. Cell Biol. 160:487-493.[Abstract/Free Full Text]

REEDQUIST, K. A., E. ROSS, E. A. KOOP, R. M. WOLTHUIS, and F. J. ZWARTKRUIS et al., 2000  The small GTPase, Rap1, mediates CD31-induced integrin adhesion. J. Cell Biol. 148:1151-1158.[Abstract/Free Full Text]

SAWADA, Y., K. NAKAMURA, K. DOI, K. TAKEDA, and K. TOBIUME et al., 2001  Rap1 is involved in cell stretching modulation of p38 but not ERK or JNK MAP kinase. J. Cell Sci. 114:1221-1227.[Abstract]

STRONACH, B. E. and N. PERRIMON, 1999  Stress signaling in Drosophila. Oncogene 18:6172-6182.[Medline]

TAKAHASHI, K., T. MATSUO, T. KATSUBE, R. UEDA, and D. YAMAMOTO, 1998  Direct binding between two PDZ domain proteins Canoe and ZO-1 and their roles in regulation of the jun N-terminal kinase pathway in Drosophila morphogenesis. Mech. Dev. 78:97-111.[Medline]

TAUTZ, D. and C. PFEIFLE, 1989  A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98:81-85.[Medline]

TEPASS, U., 1996  Crumbs, a component of the apical membrane, is required for zonula adherens formation in primary epithelia of Drosophila. Dev. Biol. 177:217-225.[Medline]

TSUKAMOTO, N., M. HATTORI, H. YANG, J. L. BOS, and N. MINATO, 1999  Rap1 GTPase-activating protein SPA-1 negatively regulates cell adhesion. J. Biol. Chem. 274:18463-18469.[Abstract/Free Full Text]

VAN AELST, L., 1998  Two-hybrid analysis of Ras-Raf interactions. Methods Mol. Biol. 84:201-222.[Medline]

VAN DEN BERGHE, N., R. H. COOL, G. HORN, and A. WITTINGHOFER, 1997  Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N). Oncogene 15:845-850.[Medline]

YORK, R. D., H. YAO, T. DILLON, C. L. ELLIG, and S. P. ECKERT et al., 1998  Rap1 mediates sustained MAP kinase activation induced by nerve growth factor. Nature 392:622-626.[Medline]

ZHU, J. J., Y. QIN, M. ZHAO, L. VAN AELST, and R. MALINOW, 2002  Ras and Rap control AMPA receptor trafficking during synaptic plasticity. Cell 110:443-455.[Medline]

ZWARTKRUIS, F. J. and J. L. BOS, 1999  Ras and Rap1: two highly related small GTPases with distinct function. Exp. Cell Res. 253:157-165.[Medline]

This article has been cited by other articles:

				Z. Xie, H. Photowala, M. E. Cahill, D. P. Srivastava, K. M. Woolfrey, C. Y. Shum, R. L. Huganir, and P. Penzes Coordination of Synaptic Adhesion with Dendritic Spine Remodeling by AF-6 and Kalirin-7 J. Neurosci., June 11, 2008; 28(24): 6079 - 6091. [Abstract] [Full Text] [PDF]

				B. Boettner and L. Van Aelst The Rap GTPase Activator Drosophila PDZ-GEF Regulates Cell Shape in Epithelial Migration and Morphogenesis Mol. Cell. Biol., November 15, 2007; 27(22): 7966 - 7980. [Abstract] [Full Text] [PDF]

				M. Lorger and K. Moelling Regulation of epithelial wound closure and intercellular adhesion by interaction of AF6 with actin cytoskeleton J. Cell Sci., August 15, 2006; 119(16): 3385 - 3398. [Abstract] [Full Text] [PDF]

				C. Laplante and L. A. Nilson Differential expression of the adhesion molecule Echinoid drives epithelial morphogenesis in Drosophila Development, August 15, 2006; 133(16): 3255 - 3264. [Abstract] [Full Text] [PDF]

				S. Huelsmann, C. Hepper, D. Marchese, C. Knoll, and R. Reuter The PDZ-GEF Dizzy regulates cell shape of migrating macrophages via Rap1 and integrins in the Drosophila embryo Development, August 1, 2006; 133(15): 2915 - 2924. [Abstract] [Full Text] [PDF]

				A. Sakurai, S. Fukuhara, A. Yamagishi, K. Sako, Y. Kamioka, M. Masuda, Y. Nakaoka, and N. Mochizuki MAGI-1 Is Required for Rap1 Activation upon Cell-Cell Contact and for Enhancement of Vascular Endothelial Cadherin-mediated Cell Adhesion Mol. Biol. Cell, February 1, 2006; 17(2): 966 - 976. [Abstract] [Full Text] [PDF]

				Z. Zhang, H. Rehmann, L. S. Price, J. Riedl, and J. L. Bos AF6 Negatively Regulates Rap1-induced Cell Adhesion J. Biol. Chem., September 30, 2005; 280(39): 33200 - 33205. [Abstract] [Full Text] [PDF]

				K. J. Mandell, B. A. Babbin, A. Nusrat, and C. A. Parkos Junctional Adhesion Molecule 1 Regulates Epithelial Cell Morphology through Effects on {beta}1 Integrins and Rap1 Activity J. Biol. Chem., March 25, 2005; 280(12): 11665 - 11674. [Abstract] [Full Text] [PDF]

				S. Fukuhara, A. Sakurai, H. Sano, A. Yamagishi, S. Somekawa, N. Takakura, Y. Saito, K. Kangawa, and N. Mochizuki Cyclic AMP Potentiates Vascular Endothelial Cadherin-Mediated Cell-Cell Contact To Enhance Endothelial Barrier Function through an Epac-Rap1 Signaling Pathway Mol. Cell. Biol., January 1, 2005; 25(1): 136 - 146. [Abstract] [Full Text] [PDF]

				W. P.-v. Berkel, M.H.G. Verheijen, E. Cuppen, M. Asahina, J. de Rooij, G. Jansen, R.H.A. Plasterk, J. L. Bos, and F.J.T. Zwartkruis Requirement of the Caenorhabditis elegans RapGEF pxf-1 and rap-1 for Epithelial Integrity Mol. Biol. Cell, January 1, 2005; 16(1): 106 - 116. [Abstract] [Full Text] [PDF]