InteractiveFly: GeneBrief

Roughened: Biological Overview | References

Gene name - Rap1 GTPase

Synonyms - Rap1, Dras3, roughened

Cytological map position 62B7-62B7

Function - signal transduction

Keywords - gastrulation, regulation of cell shape; germ-line stem cell maintenance; dorsal closure; mesoderm - hemocyte migration; Rap protein signal transduction; cell adhesion; establishment of ommatidial planar polarity; substrate-dependent cell migration, cell extension.

Symbol - Rap1

FlyBase ID: FBgn0004636

Genetic map position - chr3L:1,859,114-1,862,234

Classification - Ras_like_GTPase

Cellular location - cytoplasmic

NCBI link: EntrezGene
Rap1 orthologs: Biolitmine
Recent literature
Marada, S., Truong, A. and Ogden, S. K. (2015). The small GTPase Rap1 is a modulator of Hedgehog signaling. Dev Biol [Epub ahead of print]. PubMed ID: 26481064
During development, the evolutionarily conserved Hedgehog (Hh) morphogen provides instructional cues that influence cell fate, cell affinity and tissue morphogenesis. To do so, the Hh signaling cascade must coordinate its activity with other morphogenetic signals. This can occur through engagement of or response to effectors that do not typically function as core Hh pathway components. Given the ability of small G proteins of the Ras family to impact cell survival, differentiation, growth and adhesion, it was of interest to determine whether Hh and Ras signaling might intersect during development. Genetic modifier tests were performed in Drosophila to examine the ability of select Ras family members to influence Hh signal output, and Rap1 was identified as a positive modulator of Hh pathway activity. The results suggest that Rap1 is activated to its GTP-bound form in response to Hh ligand, and that the GTPase exchange factor C3G likely contributes to this activation. The Rap1 effector Canoe (Cno) also impacts Hh signal output, suggesting that a C3G-Rap1-Cno axis intersects the Hh pathway during tissue morphogenesis.
Yang, D.S., Roh, S. and Jeong, S. (2016). The axon guidance function of Rap1 small GTPase is independent of PlexA RasGAP activity in Drosophila. Dev Biol [Epub ahead of print]. PubMed ID: 27565025
Plexins (Plexs) comprise a large family of cell surface receptors for semaphorins (Semas) that function as evolutionarily conserved guidance molecules. GTPase activating protein (GAP) activity for Ras family small GTPases has been implicated in plexin signaling cascades through its RasGAP domain. However, little is known about how Ras family GTPases are controlled in vivo by plexin signaling. This study found that Drosophila Rap1, a member of the Ras family of GTPases, plays an important role controlling intersegmental nerve b motor axon guidance during neural development. Gain-of-function studies using dominant-negative and constitutively active forms of Rap1 indicate that Rap1 contributes to axonal growth and guidance. Genetic interaction analyses demonstrate that the Sema-1a/PlexA-mediated repulsive guidance function is regulated positively by Rap1. Furthermore, neuronal expression of mutant PlexA robustly restores defasciculation defects in PlexA null mutants when the catalytic arginine fingers of the PlexA RasGAP domain critical for GAP activity are disrupted. However, deleting the RasGAP domain abolishes the ability of PlexA to rescue the PlexA guidance phenotypes. These findings suggest that PlexA-mediated motor axon guidance is dependent on the presence of the PlexA RasGAP domain, but not on its GAP activity toward Ras family small GTPases.

Heo, K., Nahm, M., Lee, M. J., Kim, Y. E., Ki, C. S., Kim, S. H. and Lee, S. (2017). The Rap activator Gef26 regulates synaptic growth and neuronal survival via inhibition of BMP signaling. Mol Brain 10(1): 62. PubMed ID: 29282074
In Drosophila, precise regulation of BMP signaling is essential for normal synaptic growth at the larval neuromuscular junction (NMJ) and neuronal survival in the adult brain. However, the molecular mechanisms underlying fine-tuning of BMP signaling in neurons remain poorly understood. This study shows that loss of the Drosophila PDZ guanine nucleotide exchange factor Gef26 significantly increases synaptic growth at the NMJ and enhances BMP signaling in motor neurons. It was further shown that Gef26 functions upstream of Rap1 in motor neurons to restrain synaptic growth. Synaptic overgrowth in gef26 or rap1 mutants requires BMP signaling, indicating that Gef26 and Rap1 regulate synaptic growth via inhibition of BMP signaling. Gef26 is involved in the endocytic downregulation of surface expression of the BMP receptors thickveins (Tkv) and wishful thinking (Wit). Loss of Gef26 also induces progressive brain neurodegeneration through Rap1- and BMP signaling-dependent mechanisms. Taken together, these results suggest that the Gef26-Rap1 signaling pathway regulates both synaptic growth and neuronal survival by controlling BMP signaling.
Sawant, K., Chen, Y., Kotian, N., Preuss, K. M. and McDonald, J. A. (2018). Rap1 GTPase promotes coordinated collective cell migration in vivo. Mol Biol Cell: mbcE17120752. PubMed ID: 30156466
During development and in cancer, cells often move together in small to large collectives. In order to move as a unit, cells within collectives need to stay coupled together and coordinate their motility. How cell collectives remain interconnected and migratory, especially when moving through in vivo environments, is not well understood. The genetically tractable border cell group undergoes a highly polarized and cohesive cluster-type migration in the Drosophila ovary. This study reports that the small GTPase Rap1, through activation by PDZ-GEF, regulates border cell collective migration. Rap1 maintains cell contacts within the cluster, at least in part by promoting the organized distribution of E-cadherin at specific cell-cell junctions. Rap1 also restricts migratory protrusions to the front of the border cell cluster and promotes the extension of protrusions with normal dynamics. Further, Rap1 is required in the outer migratory border cells but not in the central non-migratory polar cells. Such cell specificity correlates well with the spatial distribution of the inhibitory Rapgap1 protein, which is higher in polar cells than in border cells. It is proposed that precisely regulated Rap1 activity reinforces connections between cells and polarizes the cluster, thus facilitating the coordinated collective migration of border cells.
Ou, M., Wang, S., Sun, M., An, J., Lv, H., Zeng, X., Hou, S. X. and Xie, W. (2018).. The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction. Exp Cell Res. PubMed ID: 30553967
Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. The study analyzed the role of Gef26 in synaptic development and function. Significant decreases were identified in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, the data demonstrate that Gef26-specific small G protein Rap1 works downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a betaPS integrin-mediated signaling pathway. Taken together, these findings define a novel role of Gef26 in regulating NMJ development and function.
Perez-Vale, K. Z., Yow, K. D., Johnson, R. I., Byrnes, A. E., Finegan, T. M., Slep, K. C. and Peifer, M. (2021). Multivalent interactions make adherens junction-cytoskeletal linkage robust during morphogenesis. J Cell Biol 220(12). PubMed ID: 34762121
Embryogenesis requires cells to change shape and move without disrupting epithelial integrity. This requires robust, responsive linkage between adherens junctions and the actomyosin cytoskeleton. Using Drosophila morphogenesis, this study defined molecular mechanisms mediating junction-cytoskeletal linkage and explores the role of mechanosensing. Focus was placed on the junction-cytoskeletal linker Canoe, a multidomain protein. The canoe locus was engineered to define how its domains mediate its mechanism of action. Surprising, the PDZ and FAB domains, which were thought connected junctions and F-actin, are not required for viability or mechanosensitive recruitment to junctions under tension. The FAB domain stabilizes junctions experiencing elevated force, but in its absence, most cells recover, suggesting redundant interactions. In contrast, the Rap1-binding RA domains are critical for all Cno functions and enrichment at junctions under tension. This supports a model in which junctional robustness derives from a large protein network assembled via multivalent interactions, with proteins at network nodes and some node connections more critical than others.


The establishment and maintenance of apical-basal cell polarity is critical for assembling epithelia and maintaining organ architecture. Drosophila embryos provide a superb model. In the current view, apically positioned Bazooka/Par3 is the initial polarity cue as cells form during cellularization. Bazooka then helps to position both adherens junctions and atypical protein kinase C (aPKC). Although a polarized cytoskeleton is critical for Bazooka positioning, proteins mediating this remained unknown. This study found that the small GTPase Rap1 and the actin-junctional linker Canoe/afadin are essential for polarity establishment, as both adherens junctions and Bazooka are mispositioned in their absence. Rap1 and Canoe do not simply organize the cytoskeleton, as actin and microtubules become properly polarized in their absence. Canoe can recruit Bazooka when ectopically expressed, but they do not obligatorily colocalize. Rap1 and Canoe play continuing roles in Bazooka localization during gastrulation, but other polarity cues partially restore apical Bazooka in the absence of Rap1 or Canoe. The current linear model for polarity establishment was tested. Both Bazooka and aPKC regulate Canoe localization despite being 'downstream' of Canoe. Further, Rap1, Bazooka, and aPKC, but not Canoe, regulate columnar cell shape. These data suggest that polarity establishment is regulated by a protein network rather than a linear pathway (Choi, 2013).

Polarity is a fundamental property of all cells, from polarized cell divisions in bacteria or fungi to the elaborate polarity of neurons. Among the most intensely studied forms of polarity in animal cells is epithelial apical-basal polarity. Polarity of epithelial sheets is key to their function as barriers between body compartments, and is also critical in collective cell migration and cell shape change during morphogenesis, as cytoskeletal and apical-basal polarity often go hand in hand. Loss of apical-basal polarity is a hallmark of metastasis. Significant advances have been made in defining the machinery required for cell polarity in many settings, but fundamental questions remain unanswered (Choi, 2013).

Cadherin-catenin complexes, which assemble into adherens junctions (AJs) near the apical end of the lateral cell interface, are critical polarity landmarks that define the boundary between apical and basolateral domains. Studies in C.elegans and Drosophila identified other key regulators of apical-basal polarity. In the textbook view, the apical domain is defined by the Par3/Par6/aPKC and Crumbs/Stardust(Pals1)/ PATJ complexes, while Scribble, Dlg, Lgl, and Par1 define the basolateral membrane (Choi, 2013).

Complex cross-regulatory interactions between apical and basolateral proteins maintain these mutually exclusive membrane territories. These proteins also regulate other types of polarity during morphogenesis; e.g., fly Par3 (Bazooka; Baz), aPKC, and AJ proteins are planar-polarized during fly convergent extension, thus regulating polarized cell movements (Choi, 2013).

Polarized cytoskeletal networks also play key roles in establishing and maintaining apical-basal and planar polarity. These networks are thought to be physically linked to apical junctional complexes. The earlier model suggesting that cadherin-catenin complexes link directly to actin via α-catenin is now viewed as over-simplified. Instead, different proteins are thought to mediate this connection in different tissues and at different times (Choi, 2013).

Among the linkers is Canoe (Cno)/Afadin, an actin-binding protein that binds transmembrane nectins via its PDZ domain. While originally hypothesized to be essential for cell adhesion, subsequent work supports a model in which afadin modulates adhesive and cytoskeletal machinery during cell migration in vitro (see Fournier, 2011) and the complex events of mouse gastrulation. Afadin has two N-terminal Ras association domains for which the small GTPase Rap1 is the major binding partner (Linnemann, 1999), and Afadin and Rap1 are functionally linked in both flies and mice (Boettner, 2003; Hoshino, 2005). Rap1, Cno, and the Rap1 GEF Dizzy/PDZGEF are all essential for maintaining effective linkage between AJs and the apical actomyosin cytoskeleton during apical constriction of Drosophila mesodermal cells during fly gastrulation (Sawyer, 2009; Spahn, 2012). Rap1 regulates Cno localization to the membrane (Sawyer, 2009). Cno plays a related role during convergent extension, though its role is planar polarized during this process (Sawyer, 2011). Cno also regulates collective cell migration, signaling, and oriented asymmetric divisions. The Rap1/Cno regulatory module is also important in disease, as Afadin and Rap1 are implicated in congenital disorders of the cardiovascular system (Glading, 2007) and cancer metastasis (Fournier, 2011). It remains unclear whether these diverse roles all involve junction-cytoskeletal linkage or whether some are independent functions (Choi, 2013).

The small GTPase Rap1 plays diverse cellular roles. Mammalian Rap1 isoforms are perhaps best known for regulating integrin-based cell matrix adhesion (Bos, 2005; Kim, 2011), but Rap1 also regulates cell-cell AJs in both Drosophila and mice (Kooistra, 2007; Boettner, 2009). In murine endothelial cells, for example, Rap1, its effector Krit1, and VE-cadherin form a complex that regulates endothelial cell junctions and stabilizes apical-basal polarity (Glading, 2007; Lampugnani, 2010; Liu, 2011; Choi, 2013 and references therein).

In Drosophila imaginal disc cells, Rap1 regulates the symmetric distribution of DE-cadherin (DEcad) around the apical circumference of each cell (Knox, 2002). Rap1 carries out these functions via a diverse set of effector proteins, including Krit1, TIAM, RIAM, and Cno/Afadin (Kooistra, 2007; Boettner, 2009). Thus, Rap1 and its effectors are candidate proteins for regulating interactions between AJs, polarity proteins and the cytoskeleton during polarity establishment and maintenance (Choi, 2013).

The early Drosophila embryo provides among the best models of establishing and maintaining apical-basal polarity. Flies start embryogenesis as a syncytium, with 13 rounds of nuclear division without cytokinesis. Membranes then simultaneously invaginate around each nucleus, forming ~6000 cells in a process known as cellularization. Prior to cellularization, the egg membrane is already polarized and serves as a polarity cue for underlying nuclei. This ultimately becomes the apical end of the new cells. Epithelial apical-basal polarity is initiated during cellularization. In the absence of cadherin-catenin complexes, cells form normally but then lose adhesion and polarity as gastrulation begins. These data and earlier work from cell culture suggested AJs are the initial apical cue. However, it was found that Bazooka (Baz)/Par3 acts upstream of AJs in this process. Strikingly, Baz and DEcad apically co-localize in spot AJs from cellularization onset. In the absence of Baz, DEcad loses its apical enrichment and redistributes all along the lateral membrane, while in the absence of AJ proteins, Baz remains apically localized, and a subset of cells retain residual apical-basal polarity, although cell shapes are highly abnormal. Cadherin-catenin and Baz complexes form independently before cellularization, and Baz then helps position DEcad in the apicolateral position where spot AJs will form. This placed Baz atop of the polarization network, raising the question of how it is positioned apically. Two cytoskeletal networks play important roles in initial Baz positioning (Choi, 2013).

Disrupting dynein led to Baz spreading along the lateral membrane, suggesting polarized transport along microtubules (MTs) plays a role. Depolymerizing actin also destabilized apical Baz, as did significantly overexpressing Baz, suggesting an actin-based scaffold with a saturable number of binding sites anchors Baz apically. While both actin and MTs are required for initial Baz polarization, they are not the only cues. Mislocalized Baz is re-recruited or re-stabilized apically at gastrulation onset if either initial cue is disrupted, suggesting a third cue perhaps involving aPKC/Par6 or Par1. Thus, the current model for initial establishment of apical-basal polarity involves a relatively simple pathway in which Baz is positioned apically, and then positions other apical polarity players. However, once initial polarity is established, events become more complex, with a network of mutually reinforcing and inhibitory interactions between apical and basolateral polarity complexes leading to polarity elaboration and maintenance. These were significant advances, but the proteins directing apical accumulation of Baz remained unknown. Work on apical constriction in the fly mesoderm, convergent extension during gastrulation, establishment of anteriorposterior polarity in one cell C. elegans embryos, and on apically constricting Drosophila amnioserosal cells, suggested that a complex network of interactions link AJs, the apical polarity proteins Baz and aPKC, and the actomyosin cytoskeleton. Recent work on Canoe and Rap1's roles in mesoderm apical constriction (Sawyer, 2009) and convergent elongation (Sawyer, 2011) suggested they also fit into this network. These data led to an exploration of whether Rap1 and Cno play roles in initial apical positioning of AJs and Baz and thus in the establishment and early maintenance of polarity (Choi, 2013).

In regulating polarity establishment, Rap1 and Cno could act by several possible mechanisms. Their role in AJ positioning may be solely due to their effects on Baz localization, or alternatively Rap1 and Cno may independently affect the localization of both Baz and AJs. In the latter case, Cno may directly link AJs to the apical actin scaffold, as it was suggested to act in apical constriction (Sawyer, 2009). Rap1 and Cno also clearly regulate Baz positioning. Since Baz apical positioning requires an apical actin scaffold and dynein based MT transport (Harris, 2005), whether Rap1 and Cno act indirectly by regulating cytoskeletal organization was examined. However, the data suggest this is not the case: both the MT and actomyosin cytoskeletons appear normal in mutants. Thus the most likely model is that Rap1 and Cno are required for anchoring Baz apically. Consistent with this, when Cno was ectopically localized to artificial cell-cell contacts in cultured fly cells, it was able to recruit Baz to that site. This could occur directly, for example, by Cno binding Baz, or indirectly, via unknown intermediaries. Strikingly, however, when Baz was over-expressed in cellularizing embryos, presumably saturating its apical binding sites, it accumulated basolaterally and recruited DEcad but not Cno to these ectopic sites. Thus Cno and Baz do not co-localize obligatorily. It likely that each has multiple binding partners and that when pools are limiting, as Cno may be in this latter experiment, ectopic Baz cannot recruit Cno away from a preferred binding site. Of course, it remains possible that Cno and Rap1 also regulate Baz positioning through effects on MT transport or, given Cno's apical localization, unloading at an apical docking site. It will be important to test these possibilities. As is discussed in more detail below, it will also be important to define the Cno- and Rap1-independent mechanisms that partially restore apical Baz localization after gastrulation onset (Choi, 2013).

Since Rap1 is uniformly distributed along the apical-basal axis during cellularization (Sawyer, 2009), the most likely hypothesis is that it is locally activated apically by a GEF. A number of Rap1GEFs exist, many of which are conserved between mammals and flies. Recent work from the Reuter lab demonstrated that, like Cno and Rap1 (Sawyer, 2009), the Rap1 GEF Dizzy (Dzy/PDZ-GEF) plays an important role in coordinated mesodermal apical constriction (Spahn, 2012), suggesting it is the GEF acting upstream of Cno and Rap1 in that process. They also suggest that Rap1 and Dzy help regulate establishment of AJs (Spahn, 2012). While similar in outline, their analysis of AJs differs from this one in detail, as they see strong effects on DEcad localization without similar effects on Arm. This is surprising, since these two proteins of the cadherin-catenin complex generally localize very similarly at the cortex. However, these differences aside, their data are consistent with Dzy acting with Cno and Rap1 in AJ establishment-it will be important to examine the effects of Dzy on Baz localization. It will also be important to determine how pre-existing egg membrane polarity is translated into localized Rap1 activity (Choi, 2013).

In addition to the parallel roles of Rap1 and Cno in regulating initial apical-basal polarization, this study identified a second role for Rap1 in establishing and maintaining columnar cell shape. The data suggest that this is partially or completely Cno-independent, and thus one of the many other Rap1 effectors may play a role in this process. It will be exciting to examine embryos mutant for other Rap1 effectors (Kooistra, 2007), such as Krit1/Bili, TIAM/Stilllife, RIAM/Pico, or RhoL to see if they are required for establishing columnar cell shape. baz and aPKC mutants also had defects in establishing columnar cell architecture. It is possible that each protein provides an independent mechanistic input into this process. This is consistent with the observed differences in the details of how columnar cell shape is disrupted, with Baz and aPKC primarily regulating apical cell area, while Rap1 affects cell shape at multiple apical-basal positions. A more speculative but perhaps less likely possibility is that Rap1 uses Baz and aPKC as effectors in establishing columnar cell shape. Fly Rap1 can form a complex with aPKC and Par6 (Carmena, 2011), and Rap1 acts upstream of cdc42/Par3/aPKC in regulating polarity of cultured neurons (Schwamborn, 2004; Choi, 2013 and references therein).

Having identified Rap1's direct effector(s) in regulating cell shape, it is necessary to move downstream. Based on analogies with other epithelial tissues in fly development, it is hypothesized establishing columnar cell shape involves regulating apical tension. Other small GTPases play key roles in this; e.g., Rho and cdc42 have striking and opposing roles in apical tension regulation during fly eye development. In that context, Rho acts via separate effectors to maintain AJs and apical tension-it regulates tension via Rok, Diaphanous, and ultimately myosin contractility. It will be interesting to determine whether the defects in apical cell shape in the absence of Rap1, Baz, or aPKC also reflect unbalanced contractility in different nascent cells, and which contractility regulators are involved. However, for now, this is speculative (Choi, 2013).

Previous work has suggested a linear hierarchy regulating polarity establishment, with Baz at the top, positioning AJs and aPKC (Harris, 2004, Harris, 2005). The current work extends this hierarchy, positioning Rap1 and Cno upstream of Baz in this process. However, the data further suggest that viewing polarity establishment as a linear process is significantly over-simplified. It is now known that all of the relevant players -- including the AJ proteins, Baz, Cno and aPKC -- are at the cortex in syncytial embryos, prior to cellularization and the initiation of apical-basal polarity. This places them in position to cross-regulate one another. Consistent with this, the data suggest that viewing relationships with an 'upstream-downstream' point of view misses important reciprocal interactions that occur as polarity is established. Two examples point this out most clearly. First, earlier work suggested that localization of aPKC occurs 'downstream' of Baz, as apical positioning of aPKC at gastrulation onset requires Baz function (Harris, 2005). The new data reveal that Rap1 and Cno are, in turn, 'upstream' of Baz, and thus, if things work in a strictly linear fashion, Rap1 and Cno should be 'upstream' of aPKC. However, in contrast to this simple view, this study found that precise positioning of Cno during cellularization requires aPKC - in its absence, Cno is not cleared from the apical region, and the apical-basal cables of Cno at tricellular junctions are not properly assembled. In a similar fashion, Baz, which in a linear model is 'downstream' of Cno, also regulates precise positioning of Cno during cellularization. aPKC and Baz also play important roles in Cno localization during the early polarity maintenance phase beginning at gastrulation onset. Together, these data suggest that initial positioning of proteins along the apical-basal axis involves a network of protein interactions, similar to that previously suggested to regulate polarity elaboration during the extended germband phase and beyond, as cells develop the full suite of epithelial junctions. It will now be important to define mechanisms by which aPKC and Baz act to precisely position Cno: two broad possibilities are that they act on Cno directly, or that they modulate the fine scale architecture of the actin cytoskeleton, with indirect effects on Cno. It will also be exciting to determine if other polarity determinants, like the basolateral proteins Discs Large, Scribble or Lgl, or the basolateral kinase Par1 also play roles in polarity establishment, as they do in polarity maintenance. Consistent with this possibility, recent work from the Harris lab suggests Par1 is important for the gastrulation onset rescue of Baz localization in embryos in which early cues are disrupted (McKinley, 2012). Finally, it will be interesting to identify the cues that come into play at gastrulation onset, which partially restore apical Baz localization, as part of the increasingly complex network of partially redundant regulatory cues that give polarity its robustness (Choi, 2013).

Distinct Rap1 activity states control the extent of epithelial invagination via α-Catenin

Localized cell shape change initiates epithelial folding, while neighboring cell invagination determines the final depth of an epithelial fold. The mechanism that controls the extent of invagination remains unknown. During Drosophila gastrulation, a higher number of cells undergo invagination to form the deep posterior dorsal fold, whereas far fewer cells become incorporated into the initially very similar anterior dorsal fold. A decrease in α-catenin activity causes the anterior fold to invaginate as extensively as the posterior fold. In contrast, constitutive activation of the small GTPase Rap1 restricts invagination of both dorsal folds in an α-catenin-dependent manner. Rap1 activity appears spatially modulated by Rapgap1, whose expression levels are high in the cells that flank the posterior fold but low in the anterior fold. A model is proposed whereby distinct activity states of Rap1 modulate α-catenin-dependent coupling between junctions and actin to control the extent of epithelial invagination (Wang, 2013).

This study used the dorsal fold system to investigate whether specific cellular mechanisms actively regulate the extent of epithelial invagination. α-catenin was shown to be required for the restricted invagination caused by constitutive activation of Rap1, and Rapgap1 was identified as a locally expressed modulator of Rap1 that is required for the extensive invagination of the posterior fold. These data suggest a model whereby Rap1 regulates dorsal fold invagination through an α-catenin-dependent process and establish that differential regulation of an active, specific cellular mechanism confers distinct properties to the neighboring cells to control the extent of epithelial invagination (Wang, 2013).

Genetic analysis identifies two separate functions of Rap1 during dorsal fold formation. The early function appears to be a general role required in all cells that is important for junctional positioning. This was established via examination of embryos that lack Rap1 activity, such as embryos that are produced by the germline clones of null alleles of Rap1 or dizzy, which encodes the Drosophila homolog of PDZ-GEF, a known guanine nucleotide exchange factor that activates Rap1 or embryos that overexpress a GDP-locked, dominant-negative form of Rap1, Rap1N17 (see Spahn, 2012). These embryos display normal assembly of the adherens junctions, the initial basal shift of junction positioning in the initiating cells, and attempt to form dorsal folds. Subsequently, however, the junctions relocalize to the apical surface in all dorsal cells, reversing these initial attempts of dorsal fold formation and eliminating all folding structures (Wang, 2013).

Rap1 appears to maintain the junctional positioning by maintaining the junctional levels of Bazooka. This notion is supported by the lower levels of junctional Bazooka in the Rap1 mutant embryos and by suppression of the loss-of-function phenotype of Rap1 by Bazooka overexpression, which restores the apical domain in the initiating cells and the dorsal fold structures in the Rap1 mutant embryos. Because Bazooka levels are uniform across the dorsal epithelium), this early function of Rap1 appears broadly required, independently of the levels of Rapgap1 expression, and operates in addition to Rap1's later role during epithelial invagination. The effective suppression of Rap1 loss of function following Bazooka overexpression suggests that the two separate functions of Rap1 (the maintenance of Bazooka levels and the regulation of junction-actin connection during epithelial invagination) could be decoupled, allowing comparison of the effect of loss of Rap1 function to that of constitutively active Rap1V12 (Wang, 2013).

The later, spatially regulated function of Rap1 is independent of Bazooka and is differentially modulated by the spatially restricted expression of Rapgap1. Since active Rap1 appears to act through α-catenin to inhibit invagination, it seems plausible that distinct Rap1 activity states modulate the coupling strength between junctions and actin, thereby conferring distinct properties of junctional restructuring to the neighboring cells of the anterior and posterior folds. The geometric measurements of the neighboring cells suggest a model whereby constitutively active Rap1 inhibits junctional mobility so that the size of the apical domain remains constant in the cells surrounding the anterior fold where Rapgap1 levels are low. In contrast, Rapgap1 expression modulates Rap1 activity to promote junctional mobility in the neighboring cells of the posterior fold so that their apical domain expands. In this view, both the initiation and invagination processes require active remodeling of the junctions, but differ in their underlying cellular mechanisms. During initiation, the junctional shift is induced by a modification of the epithelial apical-basal polarity as a result of the downregulation of Par-1 in the initiating cells (Wang, 2012). During invagination, since Par-1 levels do not decrease, mechanical stress might be the dominant force that causes the junctions to move in the neighboring cells (Wang, 2013).

How Rap1 modulates α-catenin-dependent junction-actin coupling remains unknown. The intensities, localization, and turnover kinetics (as measured by fluorescent recovery after photobleaching) were examined of the core junctional components (E-Cadherin and Armadillo), α-catenin, two junctional proteins that interact with both α-catenin, and actin, but no difference between the neighboring cells of the anterior and posterior folds was detected. Recent work in mammalian tissue culture cells showed that the FRET (fluorescent resonance energy transfer) intensities of an E-Cadherin tension sensor correlate with the actin-coupling states of adherens junctions (Borghi, 2012). The use of such a sensor in the living Drosophila embryo might help to reveal the difference in junction-actin coupling states between the anterior and posterior fold neighboring cells (Wang, 2013).

Recent work suggests that α-catenin undergoes a conformational change upon mechanical stretch at the cell junctions. Such conformational change could in principle relieve α-catenin from an intramolecular inhibition on actin binding, thereby increasing its affinity to, or stabilizing its interaction with, the junctional actin. Changes in α-catenin conformation thus may determine its ability to mediate the physical coupling between junctions and actin. It is of note that expression of a mutant form of α-catenin that lacks the domain that modulates its conformational change can support static junctional function, but fails to effectively rescue the loss-of-α-catenin phenotype in dynamic morphogenetic processes. It is possible that mechanical forces during morphogenesis dynamically modulate the conformational states of α-catenin, the maintenance of which may require distinct Rap1 activity states. The dynamic changes of the α-catenin conformations and the actin-coupling states of adherens junctions that they confer might be crucial for morphogenetic processes that involve extensive restructuring of cell-cell adhesion (Wang, 2013).

If Rapgap1 dictates the spatial extent of cell invagination, one simple model would envision that elevating the levels of Rapgap1 expression in the anterior region could promote anterior fold invagination. This possibility was explored using a UAS transgene to uniformly express Rapgap1 under the control of a maternal Gal4 driver. Two classes of phenotypes were observed: either a complete loss of dorsal fold formation or a limited degree of invagination similarly in both dorsal folds. The former class suggests that the level of expression may be too high to permit the normal function of Rap1, while the latter class suggests that a reversal of the expression pattern (high in the anterior fold, but low in the posterior fold) might be necessary. Attempts were made to express Rapgap1 in the cells anterior to the anterior fold using a Gal4 driver localized through the 3' UTR of the bicoid gene, and the enhancer of the Kr gene was also used to direct the expression of Rapgap1 in cells that are posterior to the anterior fold. In neither case was there an effect on anterior fold invagination. It is possible that driving extensive invagination for the anterior fold would require that Rapgap1 be expressed only in the surrounding cells of the anterior fold in a manner that mimics the endogenous pattern of Rapgap1 expression in the region of posterior fold. Currently, no cis-regulatory element or a Gal4 driver has been found that can drive gene expression in such a specific pattern. Thus, it remains unresolved whether ectopic expression in the anterior fold region would be sufficient to cause extensive invagination (Wang, 2013).

In summary, the data suggest an exciting conceptual framework in which regulated coupling between junctions and actin has a profound impact on the levels of tissue reorganization and on the cellular responses to mechanical stresses that arise during tissue reorganization. This study has defined a specific molecular pathway that produces drastically different epithelial structures from a morphogenetic process whose initiation mechanism appears similar. The regulatory principles that were unveil for Rap1 and α-catenin might be employed in other contexts of morphogenesis in which a tissue undergoes dramatic remodeling, while unperturbed tissue integrity and cell adhesion must be maintained (Wang, 2013).

Apical accumulation of the Sevenless receptor tyrosine kinase during Drosophila eye development is promoted by the small GTPase Rap1

The Ras/MAPK-signaling pathway plays pivotal roles during development of metazoans by controlling cell proliferation and cell differentiation elicited, in several instances, by receptor tyrosine kinases (RTKs). While the internal mechanism of RTK-driven Ras/MAPK signaling is well understood, far less is known regarding its interplay with other co-required signaling events involved in developmental decisions. In a genetic screen designed to identify new regulators of RTK/Ras/MAPK signaling during Drosophila eye development, the small GTPase Rap1, PDZ-GEF, and Canoe as components contributing to Ras/MAPK-mediated R7 cell differentiation. Rap1 signaling has recently been found to participate in assembling cadherin-based adherens junctions in various fly epithelial tissues. This study shows that Rap1 activity is required for the integrity of the apical domains of developing photoreceptor cells and that reduced Rap1 signaling hampers the apical accumulation of the Sevenless RTK in presumptive R7 cells. It thus appears that, in addition to its role in cell-cell adhesion, Rap1 signaling controls the partitioning of the epithelial cell membrane, which in turn influences signaling events that rely on apico-basal cell polarity (Baril, 2014).

This report describes a genetic screen in Drosophila for dominant modifiers of a CNK-dependent rough eye phenotype. Two of those modifiers, Rap1 and PDZ-GEF, were characterized to further shed light on the mechanism by which Rap1-mediated events influence photoreceptor cell development (Baril, 2014).

Given the role Connector eNhancer of KSR (CNK) plays in RTK-elicited Ras/MAPK signaling, mutations in loci encoding general components of this pathway in flies were recovered in a CNK C-terminal-dependent (CCT) screen. They correspond to Star [S; trafficking factor for the EGFR ligand Spitz, Egfr, daughter of sevenless (dos; Gab2 homolog), Son of sevenless (Sos; RasGEF), Ras85D, rl/mapk, ksr, and pointed (pnt; ETS domain transcription factor mediating MAPK activity). Mutations in two genes previously identified in RTK-dependent screens, but of unclear function, were also isolated. These are kismet [kis; chromatin remodeler] and multiple ankyrin repeats single KH domain [mask; putative RNA-binding protein (Baril, 2014).

Mutant alleles not identified in classical RTK/MAPK-dependent genetic screens, but for which a functional link to RTK signaling in flies or in other organisms had been established were also recovered. These include Btk family kinase at 29A [Btk29A; the single representative of Tec family kinases]; Delta (Dl); and three Rap1 pathway loci, PDZ-GEF, Rap1, and canoe (cno). Finally, mutations were isolated in four additional loci not previously reported to influence RTK/MAPK signaling: Pre-mRNA-processing factor 19 (Prp19), BREFeldin A sensitivity 1 (Bre1), NEM sensitive factor 2 (Nsf2), and second mitotic wave missing (swm) (Baril, 2014).

The Prp19 locus encodes a core spliceosome component. A specific role for these proteins has been unvailed in the Ras/MAPK pathway. Indeed, several splicing factors, including Prp19 and Prp8, were identified in a genome-wide RNAi screen in S2 cells for modulators of Ras-induced MAPK activation. Incidentally, a single mutant allele of Prp8 was also recovered in the CCT screen. Characterization of their implication in the pathway revealed that they specifically regulate MAPK protein levels by controlling the alternative splicing of selected introns of the mapk pre-mRNAs. It is thus likely that Prp19 alleles were recovered in the CCT screen because of their impact on endogenous MAPK levels during eye development (Baril, 2014).

The association that the last three genes (Bre1, Nsf2, and swm) might have with respect to CCT activity is less clear. Bre1 encodes a RING finger-containing E3 ligase mediating histone H2B monoubiquitination. This modification contributes to specific histone epigenetic changes such as histone H3K4 and H3K79 methylation that correlate with transcriptional activation. A role for Bre1 in Notch- and Wingless-dependent gene expression has been reported, but whether it acts similarly downstream of RTK signaling is not known. Given the concerted, yet distinct role Notch and EGFR signaling play in morphogenetic furrow progression and thereby in eye development, it could well be that, as for Dl, Bre1 was recovered primarily for its function in Notch signaling (Baril, 2014).

Nsf2 encodes an AAA ATPase involved in vesicular trafficking and synaptic vesicle release. Previous genetic studies have also associated this gene with Notch and Wingless signaling, and thus this could be the basis for the isolation of Nsf2 alleles. Alternatively, Nsf2 activity could be required in trafficking events directly involved in RTK signaling (Baril, 2014).

The swm gene [aka Su(Rux)2B encodes a novel protein that comprises a CCCH zinc finger and a RNA recognition motif. SWM localizes to the nucleus and was found to play multiple roles during Drosophila development, although its precise molecular function is not known. During eye development, Swm regulates the proliferation of undifferentiated cells by controlling their G1/S transition. In particular, third instar eye discs deprived of SWM activity are reduced in size and, as epitomized by the gene name, they lack the second mitotic wave, which corresponds to a row of cells located at a few cells distance posterior to the morphogenetic furrow that undergo a unique and synchronous round of cell division. This event increases the pool of uncommitted cells used for completing ommatidial assembly. Both Notch and EGFR signaling are essential for cell cycle progression of the uncommitted cells in the second mitotic wave, but act at distinct steps. Whether the swm alleles were recovered because of their impact on the second mitotic wave or for another role of SWM in differentiating cells remains to be investigated (Baril, 2014).

The ability of the CCT screen to identify mutations in three loci linked to Rap1 signaling strongly suggests a functional relationship between CNK and Rap1 activity. Yet, no evidence was found for physical association between CNK and Rap1 or PDZ-GEF, and thus the molecular underpinning of this relationship is currently not known. One possibility for their genetic interactions could be through their separate roles in RTK-mediated events. Rap1 signaling promotes adherens junction formation in differentiating photoreceptor cells, which contributes to their clustering. This phenomenon, in turn, is thought to enable the cells to respond to extracellular cues promoting differentiation. By lowering Rap1 activity, cohesive contacts between differentiating cells would be suboptimal and thereby would impede ommatidial assembly to some degree. In this scenario, the impact of CCT expression on photoreceptor cell differentiation would be exacerbated by heterozygous mutations in Rap1-signaling components as these would reduce the sensitivity of developing cells to differentiation cues (Baril, 2014).

Interestingly, it has been noted that loss of Rap1 activity does not prevent EGFR-induced MAPK activation per se, and thus Rap1 does not appear to work like Ras as a direct RAF activator. However, the data for this conclusion were based on small Rap1 mutant clones that were close or within the morphogenetic furrow. This work was extended by producing larger clones depleted in Rap1 or PDZ-GEF activity. These clones covered the zone where R7 cell commitment normally occurs. Markedly, it was found that reduced Rap1 signaling in this area considerably decreased MAPK activity as well as global pTyr levels and thereby mimicked the loss of RTK activity. Consistent with this, a strong impairment in R7 cell fate specification was observed (Baril, 2014).

In agreement with thes findings, Mavromatakis (2012) recently showed by genetic means that R7 cell fate specification had an absolute requirement in Rap1 activity. According to their model, R7 cell precursors sense higher Notch signaling owing to their position in the developing ommatidium, which at this stage is antagonistic to Ras/MAPK-mediated neuronal differentiation. To counteract Notch signaling, Mavromatakis (2012) proposed that presumptive R7 cells turn on two RTKs (EGFR and SEV) to produce higher MAPK activity. Intriguingly, their work suggested that Rap1 was required downstream of SEV, although they could not distinguish whether Rap1 acted through the canonical MAPK pathway or parallel to it. This was investigated and it was found Rap1 does not seem to work directly through the MAPK pathway since ectopic expression of Rap1V12 during eye development or in cultured S2 cells did not promote MAPK phosphorylation. Moreover, depletion of Rap1 or PDZ-GEF by RNAi in S2 cells had no consequence on MAPK activation induced by SEV or EGFR. Although the precise mechanism by which Rap1 influences signaling downstream of SEV remains to be delineated, the combined data suggest that Rap1 works at two distinct levels in SEV-mediated signaling, that is, upstream of SEV by modulating the apical localization of SEV and downstream of SEV by a mechanism that has yet to be characterized (Baril, 2014).

Adherens junctions form a belt-like microdomain that encircles epithelial cells apically and that play a major role in cell-cell adhesion, motility, and polarity. One of the core structural components of adherens junctions is Ecad, which is a transmembrane glycoprotein that forms Ca2+-dependent homophilic interactions between adjacent cells. The intracellular portion of Ecad is complexed to the catenins that, in turn, mediate linkage to the actomyosin cytoskeleton. Studies conducted over the past 10 years in both vertebrate and invertebrate organisms demonstrate the critical role that Rap1 signaling plays in modulating the connections of adherens junctions to the actomyosin network, which then influence cell-cell adhesion, cell shape, and cell migration (Baril, 2014).

Although the current data are consistent with this view, they also hint at a new role for Rap1 signaling that is to control apical domain formation in developing photoreceptor cells. Given that adherens junctions may act as physical barriers between apical and basolateral membrane compartments, the influence of Rap1 on adherens junction dynamics could represent the mechanism by which Rap1 exerts its effect on the apical domain compartment. A more exciting alternative would be that Rap1 activity directly controls the formation of the apical domain. Work conducted in fly embryos by Choi (2013) recently provided evidence supporting this model. Indeed, not only did that study find that Rap1 activity is essential for establishing the apico-basal polarity of cellularizing embryos, but their data also suggest that it has a direct impact on the apical localization of Bazooka, a member of the Par complex, which then orchestrates apical domain assembly. Whether Rap1 signaling has a direct influence on cell polarity during eye development is still unclear. Nonetheless, further characterization of the impact that Rap1 signaling has on apical domain formation/maintenance should reveal novel aspects by which cell compartmentalization is brought about and regulated as well as how it connects to downstream signaling events in epithelial cells (Baril, 2014).

Direct binding of Talin to Rap1 is required for cell-ECM adhesion in Drosophila

Attachment of cells to the Extracellular Matrix (ECM) via integrins is essential for animal development and tissue maintenance. The cytoplasmic protein Talin is necessary for linking integrins to the cytoskeleton and its recruitment is a key step in the assembly of the adhesion complex. However, the mechanisms that regulate Talin recruitment to sites of adhesion in vivo are still not well understood. This study shows that Talin recruitment to, and maintenance at, sites of integrin-mediated adhesion requires a direct interaction between Talin and the GTPase Rap1. A mutation that blocks the direct binding of Talin to Rap1 abolished Talin recruitment to sites of adhesion and the resulting phenotype phenocopies null alleles of Talin. Moreover, this study shows that Rap1 activity modulates Talin recruitment to sites of adhesion via its direct binding to Talin. These results identify the direct Talin-Rap1 interaction as a key in vivo mechanism for controlling integrin-mediated cell-ECM adhesion (Camp, 2018).

Although Rap1 has been known as a major regulator of integrin-based adhesion for a long time, the idea that it binds to Talin directly is relatively recent. In Drosophila Rap1 plays diverse roles, and in particular is an essential regulator of cell-cell adhesion. Flies completely lacking Rap1 die during the first stages of embryonic development and exhibit severe cellular polarity defects that prevent the development of complex tissue. Importantly, Rap1 has also been previously implicated in regulating cell-ECM adhesion in Drosophila, although the mechanisms remain elusive. Initial indications of a possible direct binding of Talin and Rap1 came from solving the structure of the vertebrate talin 1 F0 domain, which revealed that the F0 domain exhibited structural similarity to the Ras-binding site in RalGDS (Goult, 2010). Subsequent studies carried out in both Dictyostelium and mammalian cell culture confirmed this direct binding and showed that it is functionally important (Plak, 2016; Zhu, 2017). Two key functional observations have emerged from these studies: first, that the binding of Talin to Rap1 is low affinity and, second, that the binding of Talin to Rap1 regulates Talin recruitment to the membrane (Plak, 2016; Zhu, 2017). The current work confirms and builds upon these previous observations. The phenotype observed when a mutation was introduced in Talin that blocks Rap1 binding is indistinguishable from that observed in a null mutation that completely abolishes Talin function. FRAP and localization studies suggests that the strong phenotype caused by loss of direct binding of Talin to Rap1 can be explained by a disruption in the ability of the mutant Talin to be localized to and/or be maintained at sites of integrin-mediated adhesion. This result is somewhat surprising because Talin has multiple means of localizing to sites of integrin-mediated adhesion, including two integrin-binding sites, a binding site for the RAP1-binding scaffolding protein RIAM, a phosphatidylinositol 4,5-bisphosphate (PIP2) interaction domain, as well as multiple other domains that interact with other components of the adhesion complex. It thus appears that, at least in certain contexts, the interaction with Rap1, although weak in nature, is of particular importance for controlling Talin localization. Previous work has suggested that the direct interaction between Talin and Rap1 is strengthened when Rap1 is anchored to the membrane (Zhu, 2017). In this model, Rap1 localization to sites of adhesion creates a microenvironment that favours the recruitment and/or maintenance of Talin (Zhu, 2017). This model is very compatible with the current findings and helps explain earlier results that defined a role for Rap1 in regulating integrin-mediated adhesion in flies (Camp, 2018).

The direct binding of Rap1 to Talin also fits very well with earlier work on Talin autoinhibition (Ellis, 2013). Previous work showed that Talin that is unable to undergo autoinhibition localizes to the membrane independently of the presence of RIAM (Ellis, 2013). This suggested the existence of a RIAM-independent mechanism for Talin localization. Intriguingly, previous studies show that the F2F3 domain of Talin contains a conserved RIAM-binding site. The results show that although the F2F3 domain of Talin localizes to sites of adhesion, it does so less efficiently than the full Talin head domain (containing F0-F3). Furthermore, in contrast to the full-length Talin head construct, the recruitment of a construct containing only the F2F3 domain was not efficiently modulated by the presence of constitutively activated Rap1. This indicates that the main way Rap1 controls the recruitment of the Talin head domain to sites of integrin-mediated adhesion is through direct binding to Talin rather than indirect binding through RIAM. Support for these conclusions comes from studies in mice that showed that RIAM was dispensable in most tissues for Talin localization and integrin activation. Therefore, it appears that in flies, and possibly, in some tissues in mice, the direct interaction of Talin with Rap is both necessary and sufficient for recruitment and/or maintenance of Talin at sites of integrin-mediated adhesion, and that this applies to both autoinhibited and non-autoinhibited Talin (Camp, 2018).

The data suggests that a Talin mutant that is unable to bind directly to Rap1 is transcribed, translated and folds normally, but is unable to function. While a model is favored wherein the strong phenotype that was observed is due to an inability to localize and/or maintain Talin at sites of adhesion, several other alternative hypotheses cannot be discounted. For example, it could be that, in addition to disrupting the interaction of Talin with Rap1, the K17 mutation also disrupts the interaction of Talin with proteins other than Rap1. Possible candidates for such additional interactions with the Talin F0 domain are other Ras GTPases encoded by the Drosophila genome. However, the possible roles of fly GTPases were explored in the context of a genome wide analysis that analysed, among other phenotypes, disruption to integrin-based myotendinous junctions. This analysis failed to identify a role for additional Ras GTPases in integrin-mediated muscle tendon attachment. Additionally, while the experiments show direct F0-Rap1 interactions in vitro they do not address the specificity or selectivity of the interaction. The overall weak binding between Talin F0 and Rap1 makes it difficult to illustrate this interaction in vivo, which leaves the possibility of indirect binding or that additional, partially redundant, factors are involved. Furthermore, in previous work it was shown that making a 'headless' version of Talin by deleting the entire Talin head (residues 1-448), while leaving the rest of Talin intact results in a Talin protein that is completely non-functional, but that can still partially localize to sites of adhesion (x 2014). Given this result, it is curious that mutating the single K17 residue in the Talin head completely abolishes Talin localization. One possible explanation is that the headless version of Talin was expressed from a ubiquitous promoter in an exogenous rescue construct, in contrast to the CRISPR approach used in this study. Another possible explanation is that the headless Talin was tagged with GFP, and was detected using an extremely sensitive GFP-specific antibody in contrast to the Talin-specific antibody used to detect the K17 mutant Talin in the present study. Nonetheless, these results hint at the complicated network of positive and negative reinforcement cues that operate on Talin to regulate its localization to the membrane (Camp, 2018).

It remains to be fully established whether the important role of direct binding between Talin and Rap1 is conserved in vertebrates. The higher complexity of integrin-based adhesions in vertebrates provides alternative regulatory mechanisms that can mask the sort of dramatic phenotypic effects observed in disruptions of the simpler integrin-based adhesions found in the fly. Consistent with this idea is the recent analysis of mice containing a mutation in Talin designed to block the interaction of the F0 domain of Talin with Rap1 (Lagarrigue, 2018). That work shows that, at least in the context of blood platelets, the direct interaction between the F0 domain and Rap1 is not essential for integrin activation. This suggests two possible differences between the fly and vertebrate integrin-based adhesions, the first is that, in vertebrates, Talin can be recruited to the membrane by multiple, Rap1-independent, mechanisms, and the second is that, in vertebrates, Rap1 binds to Talin directly through another interaction that does not involve the F0 domain and which is not conserved in flies. Nonetheless, by showing in the fly that Rap1 binding is essential for Talin recruitment to sites of adhesion this work raises the possibility that, at least in some contexts in vertebrates, the direct binding of Talin to Rap1 may prove to be functionally important (Camp, 2018).

Discontinuities in Rap1 activity determine epithelial cell morphology within the developing wing of Drosophila

Mechanisms that govern cell-fate specification within developing epithelia have been intensely investigated, with many of the critical intercellular signaling pathways identified, and well characterized. Much less is known, however, about downstream events that drive the morphological differentiation of these cells, once their fate has been determined. In the Drosophila wing-blade epithelium, two cell types predominate: vein and intervein. After cell proliferation is complete and adhesive cell-cell contacts have been refined, the vast majority of intervein cells adopt a hexagonal morphology. Within vein territories, however, cell-shape refinement results in trapezoids. Signaling events that differentiate between vein and intervein cell fates are well understood, but the genetic pathways underlying vein/intervein cyto-architectural differences remain largely undescribed. This study shows that the Rap1 (Roughened) GTPase plays a critical role in determining cell-type-specific morphologies within the developing wing epithelium. Rap1, together with its effector Canoe, promotes symmetric distribution of the adhesion molecule DE-cadherin about the apicolateral circumference of epithelial cells. Evidence is provided that in presumptive vein tissue Rap1/Canoe activity is down-regulated, resulting in adhesive asymmetries and non-hexagonal cell morphologies. In particular Canoe levels are reduced in vein cells as they morphologically differentiate. It was also demonstrate that over-expression of Rap1 disrupts vein formation both in the developing epithelium and the adult wing blade. Therefore, vein/intervein morphological differences result, at least in part, from the patterned regulation of Rap1 activity (O'Keefe, 2012).

During the early, proliferative phase of epithelial development each cell strives to maintain adhesive contacts with its neighbors, generating, on average, a field of hexagonal-shaped cells. This uniformity is transient, however, as multiple cell types are frequently specified within a single epithelium, each with a unique function and cyto-architecture. Mechanisms must exist, therefore, for cell-type-specific shapes to emerge as these heterogeneous epithelia begin to morphologically differentiate. This study shows that in the Drosophila wing the regulation of Rap1 activity is one means by which non-hexagonal epithelial cell shapes are generated (O'Keefe, 2012).

These studies have focused on the Drosophila wing vein. Within the wing blade, veins comprise a small subset of cells, and during pupal stages of development it was shown that vein-precursor cells adopt a unique shape (trapezoidal), compared to surrounding intervein cells (hexagonal). Presumptive vein cells are first identified by high levels of Egfr activity, and previous studies have shown that Egfr signaling up-regulates the homophilic adhesion molecule DE-cad in these cells (both transcriptionally and post-translationally) (O'Keefe, 2007). High levels of cadherin generally result in apical constriction, a prominent characteristic of the adult vein. DE-cad is only one component of this morphogenetic process, however, as increased levels of DE-cad did not result in a vein-like trapezoidal shape. It was asked, therefore, what other mechanisms might determine the non-hexagonal morphology of vein precursors (O'Keefe, 2012).

In addition to elevated levels of DE-cad, another distinguishing feature of pupal vein cells is an asymmetric distribution of DE-cad about their apicolateral circumference, a phenotype most apparent when two-cell clones of ectopic veins were examined. As loss of Rap1 leads to asymmetric DE-cad (Knox, 2002; O'Keefe, 2009), it was hypothesized that Rap1 activity is down-regulated in vein precursor cells compared to surrounding intervein precursors. Consistent with this hypothesis, Rap1 over-expression dramatically disrupted pupal vein cell shape without affecting cell fate (i.e., DSRF levels). Rap1 over-expressing vein cells had more symmetric DE-Cad distributions, and did not adopt a trapezoidal morphology. This often led to morphological vein defects in the adult wing. In addition, the localization patterns of Rap1-GFP and Canoe suggested lower levels of Rap1 activity in pupal-vein precursors (compared with surrounding intervein cells). It has been previously demonstrated that the generation of Rap1 loss-of-function clones during larval stages results in vein loss (O'Keefe, 2009). Rap1 activity, therefore, plays a dual role in wing-vein formation. First, during larval and early pupal stages, Rap1 stabilizes adhesive contacts between adjacent epithelial cells, thereby facilitating Egfr signaling and maintaining vein-cell fate. Hours later, as the wing begins to differentiate, down-regulation of Rap1 activity drives the morphological changes necessary for vein formation (O'Keefe, 2012).

How does the down-regulation of Rap1 activity specifically increase DE-cad levels at vein-vein cell contacts? Rap1 recruits Cno to adherens junctions, where Cno forms a physical link between adherens junctions and the actin cytoskeleton (Sawyer, 2009). As such, Cno primarily acts as a non-enzymatic scaffolding protein, which suggests that stoichiometry between DE-cad and Cno is important. Based on immunofluorescence analysis of apicolateral cell junctions in the wing, there is a large disparity between Cno and DE-cad levels in vein cells, as Egfr/Ras signaling both up-regulates DE-cad, and down-regulates Cno. It is inferred from these data that vein cells contain far fewer adherens junction complexes that are associated with a molecule(s) of Cno (compared to intervein cells). As Cno represents the critical Rap1 effector in this context, these Cno-free adherens junction complexes would be functionally dissociated from Rap1 signaling, and free to localize in an asymmetric fashion. Relieved from spatial constraints concerning symmetry, adherens junction complexes would accumulate at vein-vein interfaces, where chances of encountering an intercellular binding partner are highest for two reasons: (1) adjacent vein cells express higher levels of DE-cad than adjacent intervein cells, and (2) adjacent vein cells contain Cno-free adherens junction complexes, which are similarly relieved from symmetry constraints (O'Keefe, 2012).

The formation of asymmetrical adhesive contacts in presumptive vein cells is coincident with changes in apical cell shape. It was asked, therefore, how changes in DE-cad localization might affect vein-cell shape, and have proposed a simple model based on examinations of a timecourse of vein differentiation. The balance between intercellular adhesion and cortical tension is a critical determinant of cell shape. Increased adhesion expands cell contacts, and cortical tension opposes this effect. The data suggest that after ~24 h APF, vein-vein cell contacts are characterized by high levels of adhesion (i.e., DE-cad) and decreased levels of cortical tension (i.e., Cno, which links adherens junctions to the actin cytoskeleton). It is hypothesized that these factors drive the expansion of vein-vein contacts at the expense of one vein-intervein cell contact, resulting in the formation of a pentagon. Real-time imaging of vein differentiation will be used in the future to test this model of morphogenesis (O'Keefe, 2012).

The Egfr/Ras and Dpp signaling pathways act in concert to specify vein-cell fate. At 12-16 h APF, Egfr/Ras activity turns on dpp expression in presumptive vein cells. After this stage of development, Dpp is required to maintain vein identity and high levels of Egfr/Ras signaling in presumptive vein cells (creating a positive feed-back loop). In contrast, these developmental signaling pathways have very different effects on cell adhesion and epithelial cell morphology. It has been shown previously that Egfr/Ras activity up-regulates DE-cad levels in vein precursors, and that it does so in a Dpp-independent fashion (O'Keefe, 2007). Results presented in this study indicate that Egfr/Ras signaling also plays the dominant role in regulating Rap1/Cno. Two-cell clones that express RasV12 phenotypically resembled Rap1 loss-of-function cells (more so than TkvQ235D clones). In addition, RasV12 down-regulated the critical Rap1 effector Cno, whereas this effect was not evident in TkvQ235D-expressing cells. As loss of Cno disassociates actin-myosin contractility from cell shape (Sawyer, 2009), RasV12 two-cell clones were less apically constricted than TkvQ235D-expressing cells. Egfr/Ras signaling is also associated with asymmetric adhesive contacts in other developmental contexts. In the Drosophila eye, for example, Egfr/Ras signaling is required in photoreceptors. Much like vein cells, photoreceptors adhere more tightly to one another than to surrounding cells. This raises the possibility that Egfr down-regulates Rap1 activity in multiple cell types following their specification, enabling them to differentiate appropriate cell shapes. Finally, it will be interesting to determine how the Egfr/Ras and Dpp signaling pathways regulate other aspects of vein-cell morphology (e.g., constriction along the apical/basal axis to generate a lumen) (O'Keefe, 2012).

In the wing, Egfr/Ras signaling does not affect Rap1/Cno activity at every developmental stage. High levels of Egfr/Ras signaling are detected in vein cells at the beginning of the third larval instar, but vein/intervein cell-shape differences are not observed before ~24 h APF. As such, the Rap1/Cno complex likely represents a pupal-specific target of Egfr signaling. This study has shown, therefore, that a single developmental signaling pathway can first determine a cell's fate, and later contribute towards its morphological differentiation. Critical to this process, therefore, are genetic and/or epigenetic factors that temporally regulate the output of Egfr/Ras signaling. In the future it will be important to identify such factors not only for the Egfr/Ras pathway, but other developmental signaling pathways as well (O'Keefe, 2012).

Finally, it is becoming increasingly clear that Rap1 affects cancer progression, often by promoting metastasis. In cancer cells, levels of Rap1 activity are typically high, which stimulates migration and metastasis by up-regulating integrin-based cell adhesion. Such is the case in pancreatic, prostate, and breast cancers. However, loss of Rap1 can also cause metastasis by down-regulating cadherin and disrupting the epithelial integrity of the tumor (e.g., ovarian and prostate cancer). Within this disease context, the Egfr/Ras and Rap1 signaling networks often interact. Most recently, Egfr activation of Rap1 has been shown to promote metastasis of human pancreatic carcinoma cells. The precise mechanisms by which Egfr/Ras signaling affects Rap1 activity (both during normal development and disease) must be deciphered, therefore, if these metastatic processes are to be understood and/or mitigated (O'Keefe, 2012).

The PDZ-GEF protein Dizzy regulates the establishment of adherens junctions required for ventral furrow formation in Drosophila

The PDZ-GEF protein Dizzy (Dzy) and its downstream GTPase Rap1 have pleiotropic roles during development of the Drosophila embryo. This study shows that maternally provided Dzy and Rap1 first function during ventral furrow formation (VFF) where they are critical to guarantee rapid apical cell constrictions. Contraction of the apical actomyosin filament system occurs independently of Dzy and Rap1, but loss of Dzy results in a delayed establishment of the apical adherens junction (AJ) belt, whereas in the absence of Rap1 only a fragmentary apical AJ belt is formed in the epithelium. The timely establishment of apical AJs appears to be essential for coupling actomyosin contractions to cell shape change and to assure completion of the ventral furrow. Immediately after VFF, the downregulation of Dzy and Rap1 is necessary to allow normal mesodermal development to continue after the epithelial-to-mesenchymal transition, as overexpression of Dzy or of constitutively active Rap1 compromises mesodermal migration and monolayer formation. It is proposes that Dzy and Rap1 are crucial factors regulating the dynamics of AJs during gastrulation (Spahn, 2012).

This study found that both the PDZ-GEF Dzy and its target Rap1 are required for ventral furrow formation (VFF) during Drosophila gastrulation. In the absence of Dzy the establishment of the circumferential adhesion belt is slowed down while in the absence of Rap1 only a fragmentary adhesion belt is formed). In the case of dzy, this slowdown in apical junction assembly translates into a slowdown of apical cell constriction, since the cytoskeleton cannot attach to membranes during early gastrulation. Thus, first actomyosin contractions do not evoke cell shape change. In the case of rap1, junction assembly is much more severely affected, and only a fragmentary apical junction belt forms during gastrulation. This results in a variable capability of mid-ventral cells to undergo constriction since only variable levels of apical AJs are available to connect to the contracting actomyosin. Dzy and Rap1 localize cortically during and after gastrulation consistent with a role in junction assembly and possibly maintenance. Levels of both proteins are diminished in the mesoderm once it has been internalized. Overexpressing Dzy or Rap1V12 in the mesoderm results in an inhibition of mesodermal spreading, possibly by keeping up DE-Cad-mediated adhesion. The findings underline the roles of the PDZ-GEF Dzy and its GTPase Rap1 as critical factors regulating the dynamics of adherens junction (AJ) formation in Drosophila gastrulation (Spahn, 2012).

Apical constriction of ventral cells is known to be a major driving force of VFF and much progress has been made in deciphering the signal cascade leading from ventral fate determinants to an assembly of a contractile actomyosin at the apices of ventral cells. Also, the importance of tight coupling between the contracting actomyosin and the cell membranes mediated by AJs has been previously highlighted (Sawyer, 2009). Although MyoII has been implicated as a downstream target of dzy during dorsal closure (Boettner, 2007), the slowdown in cell shape change during VFF seen in dzy GLC cannot be attributed to a slowdown in apical assembly of the actomyosin apparatus. Unlike what has been reported for dorsal closure, actomyosin exhibits the same relocalization to the apex of ventral cells at the end of cellularization in dzy GLC and in wild-type. Furthermore, MyoII coalesced into balls within unconstricted cells when gastrulation starts, supporting the notion of a contracting actomyosin meshwork. Coalesced MyoII within unconstricted cells has also been reported previously for ventral cells in arm, cno and rap1 GLC (Sawyer, 2009) all of which exhibit defective cell constriction. In these studies, this observation was considered an indication of contracting actomyosin that is detached from cell membranes. The current findings are consistent with this view (Spahn, 2012).

Previous work has revealed that ventral cells are not constricted by continuous contraction and that circumferential actomyosin cables do not contribute significantly to the constriction. Instead, a medially localized actomyosin meshwork is thought to reach out to make contact to AJs at the cell membranes and executes discontinuous contraction pulses to constrict the apex (Martin, 2009). The observation that apical constriction still occurs in dzy GLC, later than in wild-type, but apparently as soon as AJs are in place, are in accordance with these findings. Thus, apical constriction is not irrecoverably affected if AJs are not ready at the onset of gastrulation. Actomyosin contraction appears to take place in a dynamical and repeated pulsed fashion over the entire time-span of gastrulation allowing cells to constrict eventually, despite an initial delay in AJ formation (Spahn, 2012).

A puzzling feature of the dzy phenotype is the failure of the ventral furrow to finally close although ventral cells have undergone complete, albeit delayed, apical constriction. It is proposed that the invagination of the mesoderm has to occur within a critical time slot, which is missed in dzy GLC due to the delay in AJ establishment and, consequently, apical cell constriction. In fact, the ventral furrow of dzy GLC very much resembles the ventral furrow of a wild-type embryo 5 to 10 minutes earlier. Still, the furrow is not properly sealed in the end, less tissue moves inside and often the furrow opens up again. This supports the notion that apical constriction alone is not sufficient to internalize the ventral furrow. Computer simulations have indicated that apical constriction alone is incapable of generating a tissue invagination and have postulated ectodermal pushing as a second source of force to internalize the ventral furrow. Such a force could be exerted by turgor pressure in medio-lateral direction within the cellular blastoderm. The ventral furrow may serve as a 'predetermined breaking line', where the tissue can give in to the inherent pressure. The delay in VFF of dzy GLC leads to a temporal overlap with germband extension and PMG invagination that immediately follow the internalization of the ventral furrow in wild-type. Both processes are likely to reduce the epithelial pressure in medio-lateral dimension since they expand the epithelium in the antero-posterior dimension. Consequently, pressure might have already become too low to generate the force required to push in the mesoderm when the 'breaking line' has finally emerged. In addition, it cannot be ruled out that the ventral furrow is not properly closed and opens up again in dzy GLC because of a failure in sealing the edges of the furrow (Spahn, 2012).

In contrast to dzy GLC, only a fragmentary AJ belt is formed in rap1 GLC as DE-Cad is diffusely distributed in the membranes and shows delayed and incomplete apical accumulation. In addition, DE-Cad reveals a striking cytoplasmic mislocalization to floating particles that are seen in rap1 GLC only. Although the nature of these particles remains to be clarified, it is speculated that they represent DE-Cad rich membrane vesicles originating from the cell membrane. It has been reported earlier (Sawyer, 2009) that initial AJ assembly is unaffected in rap1 GLC, but this conclusion was based on anti-Arm staining which look unaffected in the current analysis as well. Thus, Rap1 seems to act on DE-Cad specifically to assure its proper localization. In mammalian cells regulation of DE-Cad endocytosis has long been recognized as a cellular mechanism to modulate AJs. In this context, Rap1 has been implicated in having a key role in stabilizing DE-Cad in membrane-bound aggregates as it is thought to enhance binding of DE-Cad to p120-catenin, which may serve as a cap protecting DE-Cad from being endocytosed. On the other hand, p120-catenin appears to play only a minor role in Drosophila. Despite the accordance with previous studies (Sawyer, 2009), the unaffected apical accumulation of Arm in rap1 GLC observed in this study was surprising, especially since loss of DE-Cad is reported to entail loss of Arm in various tissues. However, Arm is also involved in many other DE-Cad independent processes, e.g., acting as a signal molecule or transcription factor, so a requirement of DE-Cad for its localization does not appear coercive (Spahn, 2012).

Albeit the precise mechanism remains to be investigated, it is assumed that in the absence of maternal Rap1, confinement of DE-Cad to cell membranes and accumulation into stable apical junctions is severely compromised. Instead, only fragmentary junctions are formed whose stability may vary stochastically. Thus, AJ fragmentation may affect different cells to a different degree. As a consequence, ventral cells show a broad distribution of constriction capability ranging from complete constriction to a total failure of constriction. It may be recognized that apical constriction does not appear to be slowed down in those cells of rap1 GLC that are capable of undergoing constriction. A reason for this could be the lack of constriction in surrounding cells, so constricting cells experience considerably less opposing force from their neighbours in the epithelium. This could allow them to constrict faster and make up the inefficient actomyosin attachment in their membranes. Similarly, the lack of constriction in neighbours may allow constricting cells to constrict uniformly ('isotropically'), rather than become eccentric like wild-type cells. Due to the discontiguous actomyosin meshwork in the ventral epithelium, tension in the anteroposterior axis will be strongly reduced so constricting cells are not forced into an eccentric morphology. Indeed, previous work has shown that mid-ventral cells can undergo isotropic constriction when anteroposterior tension is disrupted by inflicting tears upon the supracellular actomyosin meshwork (Martin, 2010). Surprisingly, in spite of the large fraction of mid-ventral cells with high constriction levels, rap1 GLC do not form a ventral furrow. It is assumed that the minor fraction of unconstricted and bloated mid-ventral cells has an inhibitory influence on VFF, possibly by interrupting the 'predetermined breaking line' (Spahn, 2012).

Thus, rap1 and dzy differ qualitatively in their maternal phenotypes because loss of Dzy only delays establishment of AJs whereas loss of Rap1 additionally entails a fragmentation of the AJ belt and massive cytoplasmic mislocalization of DE-Cad. This discrepancy is not in conflict with the concept of Dzy acting exclusively via Rap1, but strongly argues in favour of Rap1 being regulated by additional GEFs besides Dzy (Spahn, 2012).

It must be emphasized that the effects on AJ assembly seen in dzy and rap1 GLC are not confined to the prospective mesoderm but occur around the entire epithelium consistent with the localization of Dzy and Rap1 in wild-type. dzy and rap1 have been recognized as 'ventral furrow mutants' because apical constriction of ventral cells is the earliest process in embryogenesis requiring a properly built apical AJ belt (Spahn, 2012).

With the apical adhesion belt being a prominent feature of ectodermal cells, internalized mesodermal cells show substantially weaker DE-Cad intensity indicating that junctions are disassembled in order to reduce cell-cell adhesion and allow mesenchymal migration. Overexpression of Dzy or Rap1V12 impairs this mesenchymal migration significantly, Rap1V12 noticeably stronger than Dzy alone. This is very plausible given that Dzy works via Rap1 which is considerably reduced in the internalized mesoderm. Migration defects upon Rap1V12 overexpression are accompanied by significantly risen relative amounts of DE-Cad in mesenchymal cells suggesting the possibility that the downregulation of Rap1 is required to allow AJs to become disassembled in the mesoderm. Accordant results have been found in the Drosophila testis where reduction of AJs can be restored to wild-type level through overexpression of constitutively active Rap1 (Wang, 2006). It remains to be seen by what mechanism AJs are disassembled in the internalized mesoderm and how the remarkably fast diminishment of Dzy and Rap1 is triggered. Conceivably, processes accompanying EMT such as mechanical alterations in the cytoskeleton could trigger degradation signals since these processes have been found to have potential signalling ability in other systems (Spahn, 2012).

As discussed above, the discrepancy between the maternal phenotypes of dzy and rap1 implies the necessity of other GEFs acting on Rap1 during gastrulation. C3G is a tempting candidate as it has been shown to interact with Rap1 in mammalian cell culture as well as in Drosophila (Dupuy, 2005; Ishimaru, 1999). Furthermore, it exhibits GEF activity on Drosophila Rap1 in vitro (Shirinian, 2010; Spahn, 2012 and references therein).

In addition to uncovering alternative activators of Rap1 it will be interesting to identify players upstream of Dzy. Despite its cyclic nucleotide binding domain there is no indication so far that Dzy is activated by cAMP signalling. However, like several proteins involved in cell polarity, Dzy bears a PDZ domain through which it possibly binds to a membrane scaffold typically involved in mediating quick linkage between signalling molecules and structural proteins. Indeed, the PDZ protein MAGI-1 has been shown to serve as a scaffold for the vertebrate homologue of Dzy and is a good candidate for a protein giving the relevant spatial cue. Unravelling the architecture of such a signalling scaffold will be key to understanding how an epithelium can be reorganized so rapidly to allow the extraordinarily fast morphogenesis of the ventral furrow (Spahn, 2012).

Rap1 acts via multiple mechanisms to position Canoe and adherens junctions and mediate apical-basal polarity establishment

Epithelial apical-basal polarity drives assembly and function of most animal tissues. Polarity initiation requires cell-cell adherens junction assembly at the apical-basolateral boundary. Defining the mechanisms underlying polarity establishment remains a key issue. Drosophila embryos provide an ideal model, as 6000 polarized cells assemble simultaneously. Current data place the actin-junctional linker Canoe (fly homolog of Afadin) at the top of the polarity hierarchy, where it directs Bazooka/Par3 and adherens junction positioning. This study defines mechanisms regulating Canoe localization/function. Spatial organization of Canoe is multifaceted, involving membrane localization, recruitment to nascent junctions and macromolecular assembly at tricellular junctions. The data suggest apical activation of the small GTPase Rap1 regulates all three events, but support multiple modes of regulation. The Rap1GEF Dizzy (PDZ-GEF) is crucial for Canoe tricellular junction enrichment but not apical retention. The Rap1-interacting RA domains of Canoe mediate adherens junction and tricellular junction recruitment but are dispensable for membrane localization. Our data also support a role for Canoe multimerization. These multifactorial inputs shape Canoe localization, correct Bazooka and adherens junction positioning, and thus apical-basal polarity. This study integrates the existing data into a new polarity establishment model (Bonello, 2018).

Apical-basal polarity establishment is a key step in animal development, driving the assembly of epithelial tissues and organs and creating the architecture that enables morphogenetic movements. Early fly embryos provide a premier model of polarity establishment. Work from many labs has defined the assembly and apical positioning of cell-cell AJs as the key initial step. Efforts then focused on defining molecular and cellular mechanisms underlying this, revealing key roles for Baz/Par3 and, more recently, the junctional actin crosslinker Cno and its upstream activator Rap1. This refocused attention on the next level of mechanistic analysis: how does Rap1 regulate Cno localization and do Rap1-independent cues also play a role? Apical Rap1 activity promotes Cno junctional recruitment at the top of the polarity hierarchy (Bonello, 2018).

The current model of apical-basal polarity establishment during fly embryogenesis suggests the key upstream step is positioning Cno at the site where AJs will form. The small GTPase Rap1 and F-actin play roles in Cno positioning. However, the mechanism by which Rap1 acts remained unclear, as Rap1 localizes all along the invaginating membrane. The new data support a model in which apical Rap1 activity plays a key role. The ability of GTP-locked constitutively active Rap1 to recruit Cno all along the basolateral membrane and the loss of Cno from the membrane induced by GDP-locked Rap1 are both consistent with this model. In the future, it would be valuable to design a Rap1 activity sensor to confirm this hypothesis. As is discussed below, it was intriguing that although the Cno RA domain plays a role in apical Cno recruitment/retention, Rap1 also influences localization of a cno mutant lacking the RA domain. Finally, it was of interest that both the GDP- and GTP-locked Rap1 mutants altered Baz localization, raising the possibility that Baz localization requires cycling of Rap1 between the active and inactive states (Bonello, 2018).

The next task was to define Rap1 activation mechanisms. Rap1 has many GEFs, including C3G, Epac, CalDAGGEF1 and PDZ-GEF. Dzy (PDZ-GEF) was the most likely candidate as it has a role 30 min later in mesoderm apical constriction, which also requires Rap1 and Cno. Strikingly, although the analyses suggest Dzy regulates Cno in later morphogenetic events such as germband extension, junctional planar polarization and segmental groove retraction, maternal/zygotic Dzy loss did not fully mimic effects of Rap1 loss on polarity establishment. Dzy loss replicated only a subset of these effects, as cortical Cno recruitment and apical restriction were unaffected. Instead, Dzy loss specifically affected Cno enrichment at TCJs, and also led to defects in columnar cell shape like those caused by Rap1 loss. Based on this, it is hypothesized that multiple GEFs regulate Rap1 activity, each directing specific aspects of cellularization and polarity establishment. Each might create temporally or spatially restricted pools of active Rap1, with different Rap1 pools mediating different effects on Cno localization. In the accompanying paper, Schmidt (2018) describes a role for the unconventional GEF ELMO-Sponge complex in regulating initial apical positioning of Cno, consistent with this hypothesis. In the future, it will be important to investigate other Rap1GEFs, such as C3G. The cortical and apical localization of Dzy that was observed during cellularization and early gastrulation are consistent with the idea that localized Dzy provides a direct input into Rap1 activation. The data also suggest that in later events in embryogenesis, Dzy and Cno work together in many events with Rap1, but that Rap1 is also likely to regulate events where it has other activators and effectors. Another alternative is apical restriction of Rap1 activation via basolateral Rap1GAPs, as occurs at other times, and this will be important to explore. The RA domain plays important roles in Cno localization and activity but CnoΔRA retains significant function (Bonello, 2018).

The N-terminus of Cno carries two RA domains that bind Rap1. Given the essential role of Rap1 in Cno localization and function, it is suspected that the RA domains would be similarly essential. Two hypotheses were tested for the mechanism by which Rap1 could act via the RA domains to regulate Cno function during polarity establishment: (1) Rap1-GTP binding to the RA domain opens a closed, autoinhibited conformation; or (2) Rap1-GTP binding to the RA domain physically recruits Cno to sites where AJs will be assembled. The data essentially rules out the first hypothesis, as it predicted that CnoΔRA recruitment to nascent AJs would be Rap1 independent. The data are largely consistent with the second hypothesis, although the effect of Rap1 on CnoΔRA localization revealed that the Rap1-RA domain interaction is not the only means of regulation (Bonello, 2018).

The data also suggest that after gastrulation onset Rap1-independent mechanisms of Cno localization come into play, as cortical localization of both wild-type Cno and CnoΔRA begin to be restored at that stage in Rap1 knockdown embryos. Although some residual Rap1 might remain, this is thought to be unlikely as it was not detected by immunoblotting at stages when Cno localization is restored. Cno can localize to the cortex during dorsal closure in embryos expressing GDP-locked Rap1, also supporting Rap1-independent mechanisms. One potential mechanism of post-gastrulation Cno recruitment is via its known interactions with AJ proteins. Before AJs assemble, Rap1 might be essential for cortical Cno recruitment, but once AJs reappear after gastrulation onset then interactions between Cno and α-Catenin or DE-cadherin might restore Cno recruitment to AJs. Both CnoWT and CnoΔRA also retained significant function in Rap1 knockdown embryos, suggesting that Cno has Rap1-independent activity, at least when expressed at elevated levels. CnoΔRA was significantly less effective than CnoWT, confirming and extending earlier work during dorsal closure. However, CnoΔRA retained significant function in Cno knockdown embryos, suggesting that the RA domain is not absolutely essential. It might facilitate some Cno/Afadin activities; for example, the RA domain of mammalian Afadin regulates interactions with p120-catenin and modulates E-cadherin endocytosis. Cno may act as a coincidence detector, with multiple simultaneous inputs regulating its positioning and that of nascent AJs (Bonello, 2018).

Several observations support the hypothesis that Cno localization responds to multiple inputs. Rap1 activity plays a key role, as during cellularization Cno cannot localize to the membrane in the absence of Rap1 or when its ability to load GTP is compromised. It seems likely that direct interactions between Rap1 and the Cno RA domain help regulate Cno localization. However, since CnoΔRA still requires Rap1 to localize correctly, this suggests additional complexity. A second Rap1 interaction site might exist in Cno outside the RA domain. Alternately, other Rap1 effectors might regulate Cno localization by different mechanisms. During cellularization, Rap1 has Cno-independent effects on apical contractility and thus columnar cell shape. Perhaps this postulated effector alters the actomyosin cytoskeleton in a way that promotes Cno binding, since intact actin is required for Cno cortical localization. However, Cno does not simply colocalize with actin or myosin, as both are most enriched at the leading edge of the invaginating membrane. Perhaps there is an apical pool of actin in a particular conformation or with particular binding partners that allow Cno to 'choose' the correct localization. Consistent with an important role for the Cno C-terminal actin-binding domain and/or intrinsically disordered linker in localization, CnoFHA-PDZ did not localize cortically in wild-type embryos, unlike CnoΔRA. Together, these data suggest a multifactorial recruitment mechanism (Bonello, 2018).

Drosophila embryos afford unmatched temporal resolution, allowing AJ morphogenesis to be followed both in the context of apical-basal polarity establishment and later in polarity maintenance. This complex process begins with the appearance of cadherin-catenin clusters, which arise independently of a Baz polarization cue. It continues through formation of mature SAJs, which coordinate with the contractile cytoskeleton to enable the first morphogenetic movements of gastrulation. These events are now viewed as involving two independent but interlocked processes: apical restriction of Cno and AJ proteins, and their assembly into higher-order multiprotein complexes. Integrating these data with previous analyses prompts the following model. Baz helps ensure that small cadherin-catenin complexes present before cellularization are recruited/retained at an apicolateral position and assembled into larger complexes containing over 1000 cadherin molecules. Cno plays an important role, helping retain Baz at the apicolateral site of SAJ assembly. The new data suggest that Rap1 activity guides two important aspects of AJ morphogenesis -- AJ protein recruitment/retention at the apicolateral interface and the specialized assembly of larger macromolecular complexes at TCJs. These data further suggest that these two events require at least two spatial cues directed by active Rap1, one acting via the Cno RA domains and one independent of that. The results with the Rap1GEF Dzy suggest these two events have different modes of regulation. Finally, the data are consistent with a Cno-driven, self-reinforcing feedback loop in which correct Cno localization can recruit more Cno to the membrane. Cno clustering is particularly prominent at TCJs. Intriguingly, recent work in mammalian cells suggests that actomyosin cables anchor end-on at TCJs and Afadin acts there to maintain trapezoidal cell shapes. Whether Cno oligomerization is intrinsic to Cno itself or is mediated by other partners is an important question for future work. These data also have potential implications for the roles of mammalian Afadin in epithelial polarity in the kidney and intestine, another avenue for further research (Bonello, 2018).

Rap1 negatively regulates the Hippo pathway to polarize directional protrusions in collective cell migration

In collective cell migration, directional protrusions orient cells in response to external cues, which requires coordinated polarity among the migrating cohort. However, the molecular mechanism has not been well defined. Drosophila border cells (BCs) migrate collectively and invade via the confined space between nurse cells, offering an in vivo model to examine how group polarity is organized. This study shows that the front/back polarity of BCs requires Rap1, hyperactivation of which disrupts cluster polarity and induces misoriented protrusions and loss of asymmetry in the actin network. Conversely, hypoactive Rap1 causes fewer protrusions and cluster spinning during migration. A forward genetic screen revealed that downregulation of the Hippo (Hpo) pathway core components hpo or mats enhances the Rap1V12-induced migration defect and misdirected protrusions. Mechanistically, association of Rap1V12 with the kinase domain of Hpo suppresses its activity, which releases Hpo signaling-mediated suppression of F-actin elongation, promoting cellular protrusions in collective cell migration (Chang, 2018).

How cells induce group polarity to form directional protrusions is a long-standing and intensively studied question in collective cell migration. In particular, which molecular mechanism induces individual cells to compete for the leading position or to communicate with other members of the migratory cluster has yet to be established. The finding that a Rap1 and Hpo complex polarizes actin protrusion provides a mechanism underlying group polarity in collective cell migration. First, the polarity of front end-enriched actin protrusions and inward contraction directed by non-muscle myosin II are continuously retained during migration. Rap1 hyperactivity disrupts the front/rear polarity of border cells (BCs), resulting in excessive misoriented protrusions. Furthermore, individual cells expressing Rap1V12 gain an advantage in protrusion extension and take the lead in the migrating cluster, whereas Rap1N17-expressing cells behave in the opposite way. As a consequence, BCs compete to move forward, making the cluster migrate in that direction. Thus, upon higher Rap1 activity, the 'winner' cell extends protrusions and moves to the front, whereas the others, having lower activity, are outcompeted and lag behind. Second, a forward genetic screen revealed that hypoactivity of Hpo signaling enhances the Rap1V12-induced migration defect and protrusion numbers in either the backward or forward direction. Most intriguingly, pull-down assays illustrate a strong physical interaction between Rap1V12 and Hpo protein, which significantly reduces Hpo activity. Suppression of Hpo signaling has long been demonstrated to cause abnormal F-actin polymerization, which, in turn, has been linked to enormous organ size in vertebrates and invertebrates. Manipulation of the actin cytoskeleton or stiffness of the extracellular matrix also affects nuclear localization of YAP (Yes-associated protein; i.e., the mammalian homolog of Yki), which supports the role of Hpo signaling in sensing the actin architecture or local environment. Therefore, the results link Rap1 to the Hpo pathway and unfold a mechanism through which group polarity and protrusions are organized by attenuating Hpo signaling activity at the leading edge of a migrating cluster (Chang, 2018).

In general, actin-based extension occurs at the front of motile cells and, subsequently, is followed by myosin II-mediated contraction, resulting in rhythmic cycles during locomotion. In external cue-directed cell movement, chemotaxis molecules bind to membrane receptors to activate Rho GTPase and its downstream effectors, which interact with the Arp2/3 complex to initiate actin polymerization. Previous studies in the social ameba Dictyostelium discoideum uncovered that, in response to chemoattractant stimulation, spatially activated Rap1 binds to RacGEF1 at the front of cells to activate Rac1-dependent actin polymerization. Accumulated evidence suggests that, during single cell migration, Rap1 is a vital part of the polarity system that accounts for the asymmetric actin cytoskeleton. However, in terms of group migration, whether Rap1 plays the same role had not been investigated until this study, in which the conserved function of Rap1 in polarizing individual cells to move during collective migration was demonstrated (Chang, 2018).

Hpo signaling was first identified as being responsible for cell growth in Drosophila, but it has since been shown to be conserved in humans and to be pivotal in various fundamental aspects of biology, including embryonic development, tissue homeostasis, and disease. Intensive genetic screens and biochemical studies have explored the complexity of the Hpo network, but the mechanism of Hpo regulation remains less understood. The Hpo pathway can be initiated by phosphorylation of the activation loop of Hpo/MST, which is accomplished by transphosphorylation by Tao-1/TAO kinase or autophosphorylation via dimerization of the SARAH domains. Active Hpo/MST2 subsequently autophosphorylates its linker domain to create docking sites for Mats/Mob1, whose interaction with Hpo/MST2 enables it to relay the kinase cascade to Warts/large tumor suppressor kinases 1/2 (LATS1/2). The current results suggest a role for Rap1 as a Hpo signaling suppressor, impairing Hpo activation by binding to the Hpo kinase domain. It would be of great interest to investigate whether a similar mechanism operates in other systems, such as cancer metastasis, and which signals/stimuli activate Rap1 in such contexts (Chang, 2018).

In Drosophila, Hpo is activated by multiple upstream scaffold proteins such as Kib, Mer, and Ex, but the screen did not reveal any of them to have genetic interaction with Rap1 in BCs. This study also examined whether Rap1V12 affects Yki transcriptional activity using a well-characterized reporter (ex-lacZ) but no alteration in BCs was observed even in combination with one hpo mutant allele. In fact, neither wild-type nor constitutively active Yki alone delayed migration. In light of this work and other independent research showing that depletion of yki does not affect BC migration, it is concluded that Rap1 functions in the Hpo pathway independent of yki to induce biased protrusions and facilitate BC migration. Interestingly, although yki has no role in normal BC migration, a strong genetic interaction was observed between Rap1V12 and wild-type or hyperactive yki, which only arose in double gain-of-function scenarios. This finding may represent a potential mechanism by which Yki/YAP participates in Rap1-mediated migration in an oncogenic context (Chang, 2018).

Over the past decade, several upstream regulators of Hpo signaling have been identified, including adhesion junction proteins, actin-binding proteins, molecules determining apical/basal/planar cell polarity, and proteins involved in cell matrix attachment. Most of these regulators are not required to regulate the core kinase activity of Hpo through Kib, Mer, or Ex. For example, loss of capping protein (which restricts actin polymerization) leads to a reduction of Hpo signaling activity and tissue outgrowth. F-actin-destabilizing treatment in NIH 3T3 cells enhances Mst1 activity, which is sufficient to stabilize p21, a key cell cycle regulator. This evidence relating to actin cytoskeleton integrity and cell morphology has led to Hpo signaling being proposed as a mechanosensor for monitoring the local environment. However, it remains unclear whether Hpo core kinase components are directly regulated by actin architecture or whether additional adaptor proteins, such as actin-binding proteins or membrane-associated proteins, are required to transduce mechanical cues. Rap1 has been implicated in E-cadherin-dependent morphogenesis and integrin-mediated cell attachment in several tissues. The results might imply that the stress generated from BC invasion through nurse cells can be transduced to interact with Rap1 through the membrane-tethering cadherin complex, the integrin/focal adhesion complex, or even a membrane-associated protein, suppressing Hpo activity and promoting protrusion (Chang, 2018).

Rap1 has been reported to interact with the mammalian Hpo, Mst1, through RapL; i.e., the downstream effector of Rap1 that regulates T lymphocyte polarization and migration. Both these latter processes require the kinase activity of Mst1, activation of which relies on recruitment of this protein to the leading edge by Rap1 and RapL. Combined with the evidence presented in this study, the association of Rap1 with Hpo appears to be an evolutionarily conserved mechanism for advancing cellular motion, but the effect of Rap1 on Hpo/MST1 varies in different contexts. This study reveals a role for Rap1; rather than acting as a positive regulator of Hpo, it inhibits Hpo suppression of the actin polymerization involved in cell cluster migration (Chang, 2018).

In summary, this study provides cellular and molecular insights into how Rap1 regulates directional protrusions to achieve collective cell movement. In BCs, the intermolecular autophosphorylation essential for Hpo activation is hampered by its association with activated Rap1, which suppresses Hpo signaling activity and, thereby, promotes cellular protrusions (Chang, 2018).

Rap1, canoe and Mbt cooperate with Bazooka to promote zonula adherens assembly in the fly photoreceptor

In Drosophila epithelial cells, apical exclusion of Bazooka/Par3 defines the position of the Zonula Adherens (ZA), which demarcates the apical and lateral membrane and allows cells to assemble into sheets. This study shows that the small GTPase Rap1, its effector AF6/Canoe (Cno) and the Cdc42-effector Pak4/Mushroom bodies tiny (Mbt), converge in regulating epithelial E-Cadherin, and Bazooka retention at the ZA. Furthermore, the results show that the localization of Rap1, Cno and Mbt at the ZA is interdependent, indicating their functions during ZA morphogenesis are interlinked. In this context, the Rap1-GEF Dizzy was found to be enriched at the ZA and the results suggest it promotes Rap1 activity during ZA morphogenesis. Altogether, it is proposed the Dizzy, Rap1/Cno pathway and Mbt converge in regulating the interface between Bazooka and AJ material to promote ZA morphogenesis (Walther, 2018).

In the pupal photoreceptor, ZA morphogenesis is orchestrated by a conserved protein network that includes Cdc42, Par6, aPKC, Baz, Crb and its binding partner Sdt, and Par1. In turn, AJ material is an essential part of the regulatory network that orchestrates polarity. Previous work has shown that Mbt regulates pupal photoreceptor development by promoting ZA morphogenesis. During this process Mbt contributes in preventing Baz from spreading to the lateral membrane, a regulation that this study found to depend in part on the phosphorylation of Arm by Mbt at S561 and S688. It is proposed that Mbt regulates photoreceptor polarity by promoting the retention of Baz at the developing ZA. Failure in ZA retention leads to Baz spreading to the lateral membrane where it is eliminated through Par1-mediated displacement. In these cells, failure to retain AJ material, including Baz, at the ZA leads to its shortening along the apical basal axis and can impact on the polarization program of the photoreceptor (Walther, 2018).

This study shows that Mbt function is linked to that of Dzy, Rap1 and Cno. First, Cno and Mbt accumulation at the ZA is interdependent, reflecting a tight coupling between the Rap1 and Cno pathway and Mbt. Second, it was found that Cno promotes Baz retention at the ZA, as cnoIR leads to shorter ZAs that can be depleted of Arm and Baz. This phenotype resembles that of mbt mutant cells and is also seen when overexpressing a version of Arm that cannot be phosphorylated by Mbt. These observations prompted a test or the hypothesis that Rap1, Cno and Mbt might function as part of a linear pathway promoting Baz retention at the ZA. In this pathway, it was reasoned that Mbt could mediate Rap1 function through Arm phosphorylation. In testing this hypothesis, it was found that this is not the case. Instead, the observation that expressing a version of Arm that mimics its constitutive phosphorylation by Mbt does not ameliorate the cnoIR phenotype suggests that Rap1, Cno, and Mbt converge in promoting Baz retention at the ZA, and cannot compensate for each other during this process. This conclusion is well supported by the finding that overexpressing cno in mbt mutant cells does not lead to an amelioration of the mbt phenotype. Third, it was found that Mbt influences the distribution of Rap1 along the apical-basal axis of the cell in that Rap1::GFP no longer accumulates preferentially at the ZA. This correlates with a loss of Dzy::GFP at the plasma membrane, raising the possibility that Mbt might regulate Rap1 through Dzy. However, the dzy phenotype is milder than that seen with Rap1 or cno, in that loss of dzy does not lead to cell delamination from the retina. This suggests that, as has been reported in the cellularizing embryo, other GEFs regulate Rap1 during epithelial morphogenesis (Walther, 2018).

An interesting aspect of the cnoIR phenotype is the defects in apical accumulation of aPKC and Crb. These defects are not observed in the dzy mutant or Rap1IR cells, indicating that Cno might function independently of Rap1 during this process. However, it is noted that while Cno was not detected at the ZA of cnoIR cells, it can still be detected in Rap1IR cells. It is therefore hypothesized that residual Cno in Rap1IR cells supports optimum aPKC and Crb accumulation at the apical membrane. In this model, Dzy, Rap1 and Cno function as part of the same pathway, which includes a function in promoting optimum apical accumulation of Crb and aPKC. Baz is required for Par complex assembly and associated aPKC and Crb recruitment at photoreceptor apical membrane. It is hypothesized that the defects in Crb and aPKC that were detect in cnoIR cells are linked to the failure in retaining Baz at the ZA, which leads to its elimination from the lateral membrane by Par1. More work will be required to understand how exactly AJ material and ZA retention of Baz influences apical membrane specification (Walther, 2018).

Rap1 and cno have been shown to regulate apical-basal polarity in the cellularizing embryo. In this model system, Rap1 and Cno regulate the apical localization of Baz and Arm, which precedes the apical recruitment of Crb. In turn, Baz influences the localization of Cno. This work indicates that similar complex regulations are at play in the pupal photoreceptor. However, unlike in the early embryo, AJ material (Arm) is absolutely required for Baz (and Par6-aPKC) accumulation or retention at the cell cortex in the developing pupal photoreceptor. A model is therefore favored whereby Mbt, Rap1 and Cno influence ZA morphogenesis primarily through regulating the interface between E-Cad or Arm, Baz and the F-actin cytoskeleton. In this model, Mbt regulates this interface both through Arm phosphorylation and cofilin-dependent regulation of F-actin, and Cno contributes to this process, at least in part, through its ability to bind to F-actin (Walther, 2018).

To probe Rap1 and Cno function during photoreceptor ZA morphogenesis, the effect of decreasing Rap1 expression on E-Cad stability was assessed. Consistent with the notion that the function of mbt and Rap1 are linked during ZA morphogenesis, it as found that, as it is the case for Mbt , Rap1 is required to stabilize E-Cad::GFP at the photoreceptor ZA. However, the mobile fraction estimated for E-Cad is much higher in Rap1IR cells than in mbtP1 null cells (evaluated at ~70% for Rap1IR and 45% for mbtP1). Together with the finding that Mbt accumulation at the ZA is decreased in Rap1IR cells, FRAP data are therefore compatible with Mbt mediating part of the function of Rap1 in promoting E-Cad stability. However, the much larger mobile fraction were estimated in the Rap1IR genotype when compared to mbtP1 photoreceptors indicates that Rap1 must also regulate E-Cad stability independently of Mbt. The longer time scale for E-Cad::GFP to recover in Rap1IR cells when compared to mbtP1 mutant cells is compatible with Rap1 functioning, in part, through promoting E-Cad delivery (Walther, 2018).

Drosophila Raf's N terminus contains a novel conserved region and can contribute to torso RTK signaling

Drosophila Raf (DRaf) contains an extended N terminus, in addition to three conserved regions (CR1-CR3); however, the function(s) of this N-terminal segment remains elusive. In this study, a novel region within Draf's N terminus that is conserved in BRaf proteins of vertebrates was identified and termed conserved region N-terminal (CRN). The N-terminal segment can play a positive role(s) in the Torso receptor tyrosine kinase pathway in vivo, and its contribution to signaling appears to be dependent on the activity of Torso receptor, suggesting this N-terminal segment can function in signal transmission. Circular dichroism analysis indicates that DRaf's N terminus (amino acids 1-117) including CRN (amino acids 19-77) is folded in vitro and has a high content of helical secondary structure as predicted by proteomics tools. In yeast two-hybrid assays, stronger interactions between DRaf's Ras binding domain (RBD) and the small GTPase Ras1, as well as Rap1, were observed when CRN and RBD sequences were linked. Together, these studies suggest that DRaf's extended N terminus may assist in its association with the upstream activators (Ras1 and Rap1) through a CRN-mediated mechanism(s) in vivo (Ding, 2010).

Amino acids 19-77) within Draf's N terminus, conserved for Raf genes of most invertebrates and BRaf genes of vertebrates, was identified and termed CRN. This conserved region has not been described by others, but potential roles for the extended N terminus have been proposed in two reports. One found that in HeLa cells, the N terminus of BRaf may mediate Raf dimerization to generate BRaf-BRaf or BRaf-CRaf complexes, and play an important regulatory role in calcium-induced BRaf activation. Another study reported that deletion of BRaf's N terminus did not affect BRaf-CRaf dimer formation. Instead, it was found that N-terminal residues appeared to facilitate interaction with HRas in vitro. In accordance with the previous study, stronger interactions between DRaf's RBD (Ras binding domain) and the small GTPase Ras1δCAAX were observed when N-terminal and RBD sequences were linked in a yeast two-hybrid analysis. This suggested that the N terminus might assist in Ras1 binding. Furthermore, the identity of specific residues in the N terminus that might participate in Ras1 binding were mapped to the CRN region (amino acids 19-77). Two known Raf motifs, RBD and CRD, are involved in Raf's interaction with Ras. This studies, and previous results using BRaf, suggest that the N-terminal residues of DRaf and BRaf proteins, particularly the CRN region, might be another element that plays a role(s) in Ras-Raf coupling (Ding, 2010).

The small GTPase Rap shares with Ras nearly identical Raf binding regions that comprise switch 1 and the lipid moiety. Rap functions as an antagonist of Ras in regulating CRaf activity, but can activate BRaf in a parallel way with Ras. Isoform-specific features of different Raf family members may explain their distinct responses to Rap. In flies, both Ras1 and Rap1 can interact with and activate DRaf. Thus, it was reasonable to test whether DRaf's N terminus including CRN might also assist in Rap1 binding. In agreement with this idea, stronger interaction between RBD and Rap1δCAAX was observed when DRaf's CRN and RBD sequences were linked in vitro, further suggesting that the N terminus may contribute to both Ras1 and Rap1 binding potentially through a CRN-mediated mechanism(s) in vivo (Ding, 2010).

No direct interaction between Ras1 or Rap1 and the isolated DRaf N-terminal segment (amino acids 1-117) was detected, or when the N terminus was linked with the Ras1/Rap1 binding-deficient RBDR174L. Thus, the contribution of DRaf's N-terminal residues to Ras1 and Rap1 binding requires the presence of RBD. It is possible that the CRN-containing N terminus may assist in Raf-Ras interaction by making RBD more accessible to Ras1 and/or in a sequential manner, subsequent to RBD-Ras1 interaction, by stabilizing the RBD-Ras1 complex. Deletion of CRN may result in conformational or structural changes that reduce Ras1 binding affinity. Structural analysis of these complexes may provide important clues and help to understand the molecular mechanism(s) by which CRN assists in Ras-Raf interaction. The computational analysis suggested conserved CRN has the propensity to form two α-helical structures (α1 and α2) and contains a putative phosphorylation motif T-S-K located in α2. In agreement, DRaf's N terminus (amino acids 1-117) was folded in vitro and had a high content of helical secondary structure. These findings may help to establish a basis for future determination of molecular structure (Ding, 2010).

Although no verified binding partner(s) for DRaf or BRaf's N terminus has been identified, it is still possible that CRN may interact with other regulatory factors in vivo, that may affect Ras or Rap binding and/or function in activation of DRaf and BRaf. If so, the conserved structural features of CRN most likely relate to these regulatory events in vivo. Site-directed mutagenesis of conserved sites/motifs could provide useful information regarding the molecular mechanism(s) of CRN's role in the activation of DRaf and BRaf (Ding, 2010).

This in vitro study of DRaf's N terminus was initiated on the basis of in vivo findings using both loss- and gain-of-function genetic assays that deletion of N-terminal residues consistently reduces DRaf's signal potential in the Torso pathway. When expressed at high levels, FL DRaf enhanced the gain-of-function effects of the torRL3 allele much more significantly than DRafδN114. In embryos from trk-/- mothers, addition of FL DRaf, but not DRAFδN114, partially restored the A8 denticle belt structure. These findings indicate that the N terminus can play a positive role(s) in Torso RTK signaling. Interestingly, the contribution of DRaf's N terminus in the Torso pathway appeared to be dependent on upstream receptor activity, suggesting its role in transmission of the signal. Together with yeast two-hybrid data it is proposed that the presence of N-terminal residues may facilitate the association of DRaf with the upstream regulators Ras1 and Rap1, thereby assisting in transmission of the RTK signal in vivo (Ding, 2010).

For instance, in the trk- background, a small amount of active GTP-Ras1 and GTP-Rap1 are likely present, mostly due to activation by residual upstream Trunk activity, the presence of Torso-like ligand, and/or the intrinsic activity of the Torso receptor. The trk1 mutation used in this analysis results in protein truncation at the last 16 amino acids. It is possible that overexpression of FL DRaf proteins in this background increases the likelihood of interaction between abundant DRaf proteins and membrane bound GTP-Ras1 or GTP-Rap1. This in turn, could elevate the RTK signal and partially restore development of the A8 denticle belt structure in some embryos. In contrast, deletion of the N terminus could destabilize Ras1-DRaf (or Rap1-DRaf) coupling or decrease the duration of interaction, resulting in reduced DRaf signal transmission. This may explain why expression of DRafδN114 failed to rescue the A8 denticle belt in embryos from trk-/- mothers (Ding, 2010).

Previously, an auto-inhibitory role had been assigned to residues compromising the first half of the DRaf protein, in addition to their functions in promoting its activity. Deletion of the N-terminal amino acids 1-272 (including the N terminus and CR1) or 1-402 (including the N terminus, CR1, and CR2) of DRaf at least partially relieved these negative effects. In this study, although removal of the N-terminal 1-114 residues did not result in constitutive DRafδN114 activity in embryos lacking the maternal Torso receptor, it is still possible that the N terminus may contribute to auto-inhibitory effects. Together with CR1 and CR2, these N-terminal residues (1-114) may help maintain DRaf's inactive conformation. If so, the N terminus might play dual roles, both positively and negatively regulating DRaf. Therefore, its contribution to signaling may be neutralized by this auto-inhibition and consequently result in a subtle in vivo effect. If so, selective mutagenesis of the 'inhibitory' motifs/sites in the N-terminal region or removal of other cofactors involved in its negative regulation may amplify signaling differences between FL DRaf and DRafδN114. Ras binding has been thought crucial to recruit Raf to the membrane and promote its RTK signaling activity. However, the Drosophila Torso pathway appears tolerant of alterations in Ras1-DRaf coupling. Draf C110 has a R174L point mutation in the RBD domain and likely comprised for Ras1 binding. The RBDR174L is Ras binding deficient in the yeast two-hybrid assay. However, tll expression patterns and cuticles of the embryos derived from mothers with Draf C110/Draf C110 germ cells were indistinguishable from those of wild-type embryos, suggesting a mechanism(s) independent of RBD-Ras1 interaction might function in recruiting DRaf to the membrane. In agreement with this model, it has been found that membrane translocation of CRaf could be mediated by its interaction with phosphatidic acid (PA) and independent of Ras binding. This PA binding site is also conserved in ARaf, BRaf, and DRaf. Thus, DrafC110 could be recruited to the cell membranes by associating with PA. Moreover, it is known that Raf's CRD participates in Ras binding through its interaction with the lipid moiety of Ras. Once at the membrane, it is also possible that the interaction between DrafC110's CRD and Ras1 could further promote its membrane attachment and result in relatively normal Torso signal production. In this study, the presence of RBD, CRD, and the potential PA binding site may be sufficient to promote DRaf's activation in Torso signaling. This may explain why at approximately endogenous wild-type protein level maternally expressed DRafδN114 is able to rescue the embryonic terminal defects of Draf11-29 mutants. Together, considering the Torso pathway's tolerance of alterations in Ras1-DRaf coupling and the minor role DRaf's N terminus plays in Ras1 binding, it is reasonable that the phenotypic consequences of removing these N-terminal residues (DRafδN114) are not great in Torso signaling. The subtle phenotypic effects of DRaf's N terminus could also be due to compensation provided by potential autoregulatory feedback or alternative redundant processes in the in vivo system. In this study, the expression of DRaf proteins at a low level appeared to sensitize the assay system. It was found that deletion of the N terminus seemed to increase the threshold of DRaf protein levels required for normal signaling. Furthermore, by adding one copy of the ectopic torRL3 allele or removing wild-type maternal Trunk activity the sensitivity of the Torso pathway was apparently increased. These allowed the embryonic terminal system to display enhanced differences between FL DRaf and DRafδN114 proteins (Ding, 2010).

Why is this N terminus with its 'subtle' functional effects conserved during evolution, and what is its biological relevance? There are numerous RTK pathways functioning in Drosophila cellular and developmental processes. In spite of the identical Ras-Raf-MEK signal cassette they share, these RTK pathways can lead to different biological responses. Previous studies indicated that such specificity might be due to the difference in the intensity and/or duration of the signal. This suggested that the magnitude of Raf signal could function as a critical determinant of biological responses. Participation of multiple DRaf elements in Ras1 or Rap1 binding could be a good strategy to modulate its activity. Normally, tight association with Ras1 or Rap1 through RBD and CRD regions is required and sufficient to initiate the activation of DRaf, while minor adjustments/regulation of interaction by the CRN region could optimize signaling potential and reduce variability. Thus, the extended N terminus including CRN may play a role(s) as one element in a multidomain effort to promote DRaf's interaction with Ras1 and Rap1, participating and assisting in regulation to reliably attain maximal signal output (Ding, 2010).

Mesoderm migration in Drosophila is a multi-step process requiring FGF signaling and integrin activity

Migration is a complex, dynamic process that has largely been studied using qualitative or static approaches. As technology has improved, it is now possible to take quantitative approaches towards understanding cell migration using in vivo imaging and tracking analyses. In this manner, a four-step model of mesoderm migration during Drosophila gastrulation was establised: (I) mesodermal tube formation, (II) collapse of the mesoderm, (III) dorsal migration and spreading and (IV) monolayer formation. The data provide evidence that these steps are temporally distinct and that each might require different chemical inputs. To support this, the role was analyzed of fibroblast growth factor (FGF) signaling, in particular the function of two Drosophila FGF ligands, Pyramus and Thisbe, during mesoderm migration. It was determined that FGF signaling through both ligands controls movements in the radial direction. Thisbe is required for the initial collapse of the mesoderm onto the ectoderm, whereas both Pyramus and Thisbe are required for monolayer formation. In addition, it was uncovered that the GTPase Rap1 regulates radial movement of cells and localization of the beta-integrin subunit, Myospheroid, which is also required for monolayer formation. These analyses suggest that distinct signals influence particular movements, since it was found that FGF signaling is involved in controlling collapse and monolayer formation but not dorsal movement, whereas integrins are required to support monolayer formation only and not earlier movements. This work demonstrates that complex cell migration is not necessarily a fluid process, but suggests instead that different types of movements are directed by distinct inputs in a stepwise manner (McMahon, 2010).

Mesoderm migration was found to be a combination of complex three-dimensional movements involving many molecular components. live imaging, coupled with quantitative analyses, is important for studying complex cell movements, as it allowed migration to be decomposed into different movement types and thus has allowed description of subtle phenotypes. First, analysis of the directional movements of mesoderm cells within wild-type embryos was extended, focusing on the temporal sequences of events. Cells were found follow a sequential and distinct set of trajectories: movement in the radial direction (tube collapse: -5 to 15 minutes, 0=onset of germband elongation), followed by movement in the angular direction (dorsal migration: 15 to 75 minutes) and ending with small intercalation movements in the radial direction (monolayer formation: 75 to 110 minutes). These movements appear temporally distinct (i.e. stepwise), and thus molecular signals controlling each process were sought (McMahon, 2010).

Which mesoderm movements were FGF-dependent were investigated and, in particular, either Ths- or Pyr-dependent. The interaction between Htl and its two ligands provides a simpler system relative to vertebrates (which exhibit over 120 receptor-ligand interactions) in which to study how and why multiple FGF ligands interact with the same receptor. Previously, it was found that FGF signaling via the Htl FGFR controls collapse of the mesodermal tube but not dorsal-directed spreading (McMahon, 2008). This study demonstrated that FGF signaling is also required for monolayer formation. In addition, distinct, non-redundant roles were defined for the FGF ligands: Ths (but not Pyr) is required for collapse of the mesodermal tube, whereas both Pyr and Ths are required for proper intercalation of mesoderm cells after dorsal spreading (McMahon, 2010).

This analysis raises questions about ligand choice during collapse and monolayer formation. Within the mesodermal tube, cells at the top require a long-range signal in order to orient towards the ectoderm during tube collapse, whereas the signals controlling intercalation during monolayer formation can be of shorter range. It is suggested that the ligands have different activities that are appropriately tuned for these processes. In fact, recent studies of the functional domains of these proteins suggest that Ths has a longer range of action than Pyr, in agreement with the analysis that Pyr does not support tube collapse, but does have a hand in monolayer formation (McMahon, 2010).

This study has demonstrated that Rap1 mutants have a similar mesoderm phenotype to the FGFR htl mutant, with defects in collapse and monolayer formation. It was not possible to establish whether Rap1 acts downstream of FGF signaling, as the complete loss of Mys in Rap1 mutants is more severe than the patchy expression of Mys seen in htl mutants. Therefore, Rap1 could be working in parallel to or downstream of FGF signaling during mesoderm migration. Rap1 has been implicated in several morphogenetic events during Drosophila gastrulation and probably interacts with many different signaling pathways. Further study of Rap1, along with other GTPases, will shed light onto their role during mesoderm migration, how they interact with one another and what signaling pathways control them (McMahon, 2010).

Focus was placed on the more specific phenotype of mys mutants, as its localization is affected in htl mutants and it exhibits a monolayer defect that is similar to pyr and ths mutants. Integrins are important for cell adhesion, so it is not surprising that cells fail to make stable contact with the ectoderm through intercalation in mys mutants. However, some cells do contribute to monolayer formation in the absence of Mys, implying that other adhesion molecules are involved in maintaining contact between the mesoderm and ectoderm. These other adhesion molecules might be activated downstream of FGF signaling as the htl mutant monolayer phenotype is more severe than the mys mutant. Discovering the downstream targets of Htl, which might regulate cell adhesion properties, will help to shed light on the mechanisms supporting collapse of the mesodermal tube (which is not dependent on Mys) and monolayer formation (which is Mys-dependent) (McMahon, 2010).

Cell protrusions, such as filopodia, are important for sensing chemoattractants and polarizing movement during migration. Previous studies have focused on protrusive activity at the leading edge during mesoderm migration in Drosophila and shown that these protrusions are FGF-dependent. In this study, it was found that protrusions exist in all mesoderm cells, not just the leading edge, and that these protrusions also extend into the ectoderm (McMahon, 2010).

The study demonstrates that FGF signaling, as well as integrin activity, is required to support protrusive activity into the ectoderm; this is a potential mechanism by which FGF signaling and Mys could control movement toward the ectoderm during monolayer formation. The function of protrusions at the leading edge remains unclear, as they appear to be reduced in pyr and mys mutants, but migration in the dorsal direction still occurs in both mutant backgrounds. One interpretation is that FGF and Mys are important for generalized protrusive activity and that extensive protrusions are required for intercalation but not dorsal migration (McMahon, 2010).

Based on this study, it is proposed that mesoderm migration is a stepwise process, with each event requiring different molecular cues to achieve collective migration. Invagination of the mesoderm is the first step in this process and is dependent on Snail, Twist, Concertina, Fog and several other genes. Next, collapse of the mesoderm tube onto the ectoderm requires Htl activation via Ths. Rap1 might be involved in this process as well but the phenotype of Rap1 mutants is complex and it is unclear which phenotypes are primary defects (McMahon, 2010).

Following collapse, mesoderm cells spread dorsally by an unknown mechanism. Dorsal migration is unaffected in pyr and ths mutants and occurs in all cells that contact the ectoderm in htl mutants, implying that FGF signaling is, at most, indirectly involved in this step owing to the earlier tube collapse defect (McMahon, 2008). Whether dorsal migration requires chemoattractive signals or whether the cells simply move in this direction because it is the area of least resistance remains unclear (McMahon, 2010).

Finally, after dorsal spreading is complete, any remaining cells not contacting the ectoderm intercalate to form a monolayer. This process is controlled by a combination of both Pyr and Ths interacting through Htl and also by Rap1 and Mys. In other systems, intercalation can lead to changes in the properties of the cell collective, for instance, lengthening of a body plan. However, this study has shown that dorsal migration and spreading are not a result of intercalation, as intercalation occurs after spreading has finished (McMahon, 2010).

Coordination of these signals to control collective migration enables the mesoderm to form a symmetrical structure, which is essential for embryo survival. This model begins to address the question of how hundreds of cells move in concerted fashion and is relevant for a generalized understanding of embryogenesis and organogenesis. It was found that mesoderm migration is accomplished through sequential movements in different directions, implying that collective migration might be best achieved by distinct phases of movement (McMahon, 2010).

RhoL controls invasion and Rap1 localization during immune cell transmigration in Drosophila

Human immune cells have to penetrate an endothelial barrier during their beneficial pursuit of infection and their destructive infiltration of tissues in autoimmune diseases. This transmigration requires Rap1 GTPase to activate integrin affinity. A new model system for this process has been defined by demonstrating, with live imaging and genetics, that during embryonic development Drosophila melanogaster immune cells penetrate an epithelial, Drosophila E-cadherin (DE-cadherin)-based tissue barrier. A mutant in RhoL, a GTPase homologue that is specifically expressed in haemocytes, blocks this invasive step but not other aspects of guided migration. RhoL mediates integrin adhesion caused by Drosophila Rap1 overexpression and moves Rap1 away from a concentration in the cytoplasm to the leading edge during invasive migration. These findings indicate that a programmed migratory step during Drosophila development bears striking molecular similarities to vertebrate immune cell transmigration during inflammation, and identify RhoL as a new regulator of invasion, adhesion and Rap1 localization. This work establishes the utility of Drosophila for identifying novel components of immune cell transmigration and for understanding the in vivo interplay of immune cells with the barriers they penetrate (Siekhaus, 2010).

The Rap1 guanine nucleotide exchange factor C3G is required for preservation of larval muscle integrity in Drosophila melanogaster

C3G is a guanine nucleotide exchange factor (GEF) and modulator of small G-protein activity, which primarily acts on members of the Rap GTPase subfamily. Via promotion of the active GTP bound conformation of target GTPases, C3G has been implicated in the regulation of multiple cellular and developmental events including proliferation, differentiation and apoptosis. The Drosophila C3G orthologue exhibits a domain organization similar to that of vertebrate C3G. Through deletion of the C3G locus, it was observed loss of C3G causes semi-lethality, and that escaping adult flies are characterized by a reduction in lifespan and general fitness. In situ hybridization reveals C3G expression in the developing embryonic somatic and visceral muscles, and indeed analysis of C3G mutants suggests essential functions of C3G for normal body wall muscle development during larval stages. C3G mutants display abnormal muscle morphology and attachment, as well as failure to properly localize betaPS integrins to muscle attachment sites. Moreover, this study shows that C3G stimulates guanine nucleotide exchange on Drosophila Rap GTPases in vitro. Taken together, it is concluded that Drosophila C3G is a Rap1-specific GEF with important functions in maintaining muscle integrity during larval stages (Shirinian, 2010).

Rap1 maintains adhesion between cells to affect Egfr signaling and planar cell polarity in Drosophila

The small GTPase Rap1 affects cell adhesion and cell motility in numerous developmental contexts. Loss of Rap1 in the Drosophila wing epithelium disrupts adherens junction localization, causing mutant cells to disperse, and dramatically alters epithelial cell shape. While the adhesive consequences of Rap1 inactivation have been well described in this system, the effects on cell signaling, cell fate specification, and tissue differentiation are not known. This study demonstrates that Egfr-dependent cell types are lost from Rap1 mutant tissue as an indirect consequence of DE-cadherin mislocalization. Cells lacking Rap1 in the developing wing and eye are capable of responding to an Egfr signal, indicating that Rap1 is not required for Egfr/Ras/MAPK signal transduction. Instead, Rap1 regulates adhesive contacts necessary for maintenance of Egfr signaling between cells, and differentiation of wing veins and photoreceptors. Rap1 is also necessary for planar cell polarity in these tissues. Wing hair alignment and ommatidial rotation, functional readouts of planar cell polarity in the wing and eye respectively, are both affected in Rap1 mutant tissue. Finally, this study shows that Rap1 acts through the effector Canoe to regulate these developmental processes (O'Keefe, 2009).

The Rap GTPase activator Drosophila PDZ-GEF regulates cell shape in epithelial migration and morphogenesis

Epithelial morphogenesis is characterized by an exquisite control of cell shape and position. Progression through dorsal closure in Drosophila gastrulation depends on the ability of Rap1 GTPase to signal through the adherens junctional multidomain protein Canoe. This study provides genetic evidence that epithelial Rap activation and Canoe effector usage are conferred by the Drosophila PDZ-GEF (dPDZ-GEF) exchange factor. dPDZ-GEF/Rap/Canoe signaling modulates cell shape and apicolateral cell constriction in embryonic and wing disc epithelia. In dPDZ-GEF mutant embryos with strong dorsal closure defects, cells in the lateral ectoderm fail to properly elongate. Postembryonic dPDZ-GEF mutant cells generated in mosaic tissue display a striking extension of lateral cell perimeters in the proximity of junctional complexes, suggesting a loss of normal cell contractility. Furthermore, the data indicate that dPDZ-GEF signaling is linked to myosin II function. Both dPDZ-GEF and cno show strong genetic interactions with the myosin II-encoding gene, and myosin II distribution is severely perturbed in epithelia of both mutants. These findings provide the first insight into the molecular machinery targeted by Rap signaling to modulate epithelial plasticity. It is proposed that dPDZ-GEF-dependent signaling functions as a rheostat linking Rap activity to the regulation of cell shape in epithelial morphogenesis at different developmental stages (Boettner, 2007).

In developing tissues, Rap has been found to promote various morphogenetic processes, ranging from epithelial migration and invagination in embryogenesis to the maintenance of epithelial integrity in proliferating tissues at later stages. However, the mechanisms by which Rap is regulated and mediates its effects in morphogenetic episodes remain poorly understood. This report delineates a pathway in which the Drosophila GEF dPDZ-GEF links Rap activity to MyoII and the regulation of lateral contractility and cell shape in different epithelial morphogenetic episodes (Boettner, 2007).

This study identified dPDZ-GEF as a putative activator of Rap GTPases in a yeast two hybrid (YTH) screen and subsequently demonstrated that it specifically associates with Rap, but not Ras, GTPases. PDZ-GEF is highly conserved among metazoans, suggesting that it might serve common physiological roles. dPDZ-GEF was found to be highly expressed in epithelial tissues involved in embryonic dorsal closure (DC), and, importantly, the data revealed that it functions as an activator of Rap1/Cno signaling in this process. First, as in the case of Rap1 and cno, loss of zygotic dPDZ-GEF function is associated with an ectodermal failure, which is manifested by dorsal-open phenotypes. Eliminating both zygotic and maternal dPDZ-GEF elevates the frequency of late gastrulation defects. Second, the genetic analysis places dPDZ-GEF upstream of the Rap/Cno GTPase/effector complex, as both Rap1 and Cno were able to rescue the dPDZ-GEF LOF phenotype to a large extent. Third, all three proteins show an overlapping localization at AJs in ectodermal cells involved in DC. Thus, these findings demonstrate that dPDZ-GEF serves as a Rap1/Cno activator to promote late epithelial gastrulation movements. In support of a conserved role of dPDZ-GEF in epithelial morphogenesis, studies with C. elegans demonstrated that pxf-1, the dPDZ-GEF homolog, is vital for epithelial integrity. pxf-1 mutant animals often are confronted with hypodermal malfunctions; the underlying cellular basis of these defects, however, remains to be elucidated (Boettner, 2007).

Epithelial migration processes often entail striking alterations in cell shape, and much effort has been devoted to unraveling the underlying cellular and molecular mechanisms. This study highlights that dPDZ-GEF as a Rap activator adjusts cell shape to the demands of morphogenetic movements and imaginal disc morphogenesis. It was observed that dPDZ-GEF mutant embryos involved in DC often exhibit bunched regions in their leading edge and an incompetence of ectodermal cells to elongate dorsally. These phenotypes also characterize embryos that either overexpress DN Rap1 or are mutant for cno. Thus, signaling through dPDZ-GEF, Rap, and Cno (1) is vital for the organization of a coherently moving leading edge and (2) enables the typical dorsoventral stretching of lateral ectodermal cell sheets. These studies also unveiled a requirement for dPDZ-GEF for the adjustment of epithelial cell shape in the differentiation program of the wing imaginal disc. It was found that a dPDZ-GEF LOF situation generated in a clonal analysis of mosaic wing discs is associated with a decline in apicolateral contractility in the vicinity of junctional complexes. Loss of contractility, as visualized by a widening of apicolateral circumferences, is coupled to a partially compensating gain of contractility in adjacent wild-type tissue. Wild-type cells in close proximity to mutant clones display smaller apicolateral circumferences. Interestingly, overexpression of dPDZ-GEF in restricted areas of the wing disc causes contractile aberrations. When ectopically expressed in the posterior compartment, dPDZ-GEF leads to a loss of apicolateral contractility in cells lining the A/P boundary. Thus, both gain and loss of dPDZ-GEF function compromise normal contractile strength and result in aberrant adult tissue formation. These observations suggest that a finely tuned level of Rap activation is crucial for normal cellular and organismal development to occur. Tight requirements for activation of small GTPases in vivo have been documented previously, e.g., for Rho GTPases and their function in axon guidance. Importantly, this study found in genetic modification experiments that reduced or enhanced dPDZ-GEF activity in the developing wing can be rescued by ectopic Rap1 or lowered cno doses, respectively, suggesting that signaling through the dPDZ-GEF/Rap/Cno module at least partially controls disc morphogenesis. This, together with the vital cooperative roles of all three genes in embryonic cell sheet migration, corroborates the reiterative function of dPDZ-GEF/Rap/Cno signaling during epithelial development (Boettner, 2007).

What are the mechanisms that translate Rap signaling downstream of dPDZ-GEF into the modulation of cell shape? This analysis of dPDZ-GEF LOF situations during gastrulation and wing disc morphogenesis showed that junctional integrity is not corrupted. Both AJ and SJ belts around the apicolateral circumference are seamlessly maintained in dPDZ-GEF LOF tissue. However, the data support a role for the MyoII heavy chain, the product of the zip gene, as an effector. MyoII assembly and disassembly in migrating cells and tissue homeostasis are tightly balanced processes. In epithelial cells, MyoII localizes to cell-cell junctional complexes and is essential for establishing and maintaining intercellular adhesion and tension. This study found that the decline in apicolateral constriction associated with dPDZ-GEF LOF in mitotic clones in the wing disc epithelium is accompanied by a less compact MyoII localization and that adjacent constricted wild-type cells display overassembled MyoII, which concentrates in ectopic focal structures. Also, in the DC paradigm, dPDZ-GEF and cno mutant embryos that are involved in DC exhibited failures of leading-edge cells to properly assemble MyoII. The abundant MyoII localization at the leading edge that characterizes wild-type embryos during DC is significantly diminished, and the bars-on-a-string-like MyoII distribution is lost in these mutants. In particular, regions of the leading edge adjacent to the bunched segments retain only minimal amounts of assembled MyoII. These observations strongly suggest that loss of MyoII control at the leading edge is contributing to the bunching phenotype observed in dPDZ-GEF and cno mutants. Consistent with a spatiotemporal regulation of MyoII in distinct regions of the leading edge are elegant life-imaging studies undertaken with embryos undergoing DC. Dynamic cycles of MyoII-dependent contraction and relaxation occur that are limited to smaller regions within the leading edge during the migration process (Boettner, 2007).

In further support of the notion that dPDZ-GEF signaling acts on MyoII, evidence was obtained that dPDZ-GEF and cno genetically interact with zip in late gastrulation and, moreover, that cno is genetically linked to zip in wing morphogenesis. In particular, the data show a strong enhancement of dorsal-open frequencies in embryos that are double transheterozygous for hypomorphic combinations of zip and either dPDZ-GEF or cno. Combined mutations at the zip and cno loci were also found to cause a malformation of wings. Together, these findings imply that signaling through the dPDZ-GEF/Rap/Cno module is required for MyoII function at different stages of epithelial development. Future experimentation will be required to determine the precise biochemical link between this module and MyoII regulation. Of note, a recent study demonstrated that the mammalian Cno homolog, AF-6/Afadin, in a two-dimensional tissue culture system moves together with MyoII at the edge of wounds induced by laser ablation. At the onset of wound closure, a subpopulation of MyoII resides apically in the lateral membranes of cells lining the wound. However, when closure progresses into advanced stages, MyoII, together with AF-6/Afadin, migrates basalward to constrict both the wound perimeter and the apicobasal membranes facing the wound (Boettner, 2007).

These data support a model in which dPDZ-GEF, through Rap activation and MyoII regulation, contributes to the adjustment of lateral cell contractility in epithelial cells of the embryo and the developing wing. In a previous study, the analysis of Rap1 mutant clones in the wing imaginal disc revealed a direct effect of Rap1 on the reorganization of AJs at the end of cytokinesis, where resealing of their belts has to occur between daughter cells. Since the data showed that AJ integrity is unperturbed in clones comprised of dPDZ-GEF LOF cells, it is surmised that dPDZ-GEF either is not relevant for Rap1 activation in the reconstitution of a seamless AJ belt during cytokinesis or is compensated for by a still-unknown factor conferring the necessary exchange activity. In contrast, the apicolateral constriction defects detected as a consequence of clonal loss of dPDZ-GEF function so far have not been described for Rap1 mutant clones in the same scenario. It is presumed either that they have escaped scrutiny or, more likely, that Rap1 acts redundantly with its close homolog Rap2l in adjusting apicolateral constriction, while the reorganization of AJs during cytokinesis relies solely on Rap1. In fact, Rap1 and Rap2l have been shown to compensate for each other in the male stem cell niche. In this context, both Rap proteins cooperate downstream of dPDZ-GEF to anchor germ line stem cells to their niche. In future experiments, it is planned to generate Rap1 and Rap2l mitotic clones in parallel and to examine and compare their effects on cell shape and contractility (Boettner, 2007).

A picture is emerging in which specialized GEFs activate Rap GTPases and selective effectors in different morphogenetic scenarios and cellular processes. For example, Rap1 signaling has been implicated in cell/extracellular matrix-dependent force transduction at focal adhesion sites of cultured cells. In this scenario, Rap1 is regulated by an Src/p130Cas/C3G-triggered mechanism. Also, apical constriction during neural tube closure in the Xenopus blastula has been demonstrated to depend on Rap1 function downstream of the Shroom protein; however, the relevant GEF in this scenario remains to be identified. The notion that Rap activation in distinct developmental processes is specified by dedicated GEFs also suggests that Rap effectors are selected in order to fulfill pathway requirements. In light of this, dPDZ-GEF and Rap1 have been implicated in the regulation of mitogen-activated protein kinase activity during differentiation of the Drosophila compound eye, and another reported that D-Raf relays a signal from Rap to mitogen-activated protein kinase in Torso-receptor-dependent terminal differentiation of the early Drosophila embryo. Together, these findings suggest the possibility that dPDZ-GEF could trigger the activation of the Rap/D-Raf pathway to regulate certain differentiation processes. The data reveal a novel function for dPDZ-GEF as an activator of Rap in the implementation of epithelial cell shape changes required for sheet migration and homeostatic cell shape maintenance in the genesis of the wing imaginal disc epithelium. Evidence is provided that Cno functions as a relevant effector of Rap downstream of dPDZ-GEF in these events and that the dPDZ-GEF/Rap/Cno module is connected to the regulation of MyoII and the generation and modulation of appropriate lateral cell contractility. Thus, these findings have unveiled a pathway linking the Rap activator dPDZ-GEF to MyoII and the regulation of lateral contractility and cell shape in epithelium migration and homeostasis. Further elucidation of dPDZ-GEF-interacting proteins and the molecular underpinnings of MyoII regulation downstream of this module in epithelial cells will be key to understanding these aspects of tissue morphogenesis (Boettner, 2007).

The PDZ-GEF dizzy regulates cell shape of migrating macrophages via Rap1 and integrins in the Drosophila embryo

In Drosophila embryos, macrophages originate from the cephalic mesoderm and perform a complex migration throughout the entire embryo. The molecular mechanisms regulating this cell migration remain largely unknown. This study identified the Drosophila PDZ G-nucleotide exchange factor (PDZ-GEF) Dizzy as a component essential for normal macrophage migration. In mutants lacking Dizzy, macrophages have smaller cellular protrusions, and their migration is slowed down significantly. This phenotype appears to be cell-autonomous, as it is also observed in embryos with a dsRNA-induced reduction of dizzy function in macrophages. In a complementary fashion, macrophages overexpressing Dizzy are vastly extended and form very long protrusions. These cell shape changes depend on the function of the small GTPase Rap1: in rap1 mutants, Dizzy is unable to induce the large protrusions. Furthermore, forced expression of a dominant-active form of Rap1, but not of the wild-type form, induces similar cell shape changes as Dizzy does overexpression. These findings suggest that Dizzy acts through Rap1. It is further proposed that integrin-dependent adhesion is a Rap1-mediated target of Dizzy activity: in integrin mutants, neither Dizzy nor Rap1 can induce cell shape changes in macrophages. These data provide the first link between a PDZ-GEF, the corresponding small GTPase and integrin-dependent cell adhesion during cell migration in embryonic development (Huelsmann, 2006).

Rap-GEF signaling controls stem cell anchoring to their niche through regulating DE-cadherin-mediated cell adhesion in the Drosophila testis

Stem cells will undergo self-renewal to produce new stem cells if they are maintained in their niches. The regulatory mechanisms that recruit and maintain stem cells in their niches are not well understood. In Drosophila testes, a group of 12 nondividing somatic cells, called the hub, identifies the stem cell niche by producing the growth factor Unpaired (Upd). This study shows that Rap-GEF/Rap signaling controls stem cell anchoring to the niche through regulating DE-cadherin-mediated cell adhesion. Loss of function of a Drosophila Rap-GEF (Gef26) results in loss of both germline and somatic stem cells. The Gef26 mutation specifically impairs adherens junctions at the hub-stem cell interface, which results in the stem cells 'drifting away' from the niche and losing stem cell identity. Thus, the Rap signaling/E-cadherin pathway may represent one mechanism that regulates polarized niche formation and stem cell anchoring (Wang, 2006).

Novel Rap1 dominant-negative mutants interfere selectively with C3G and Epac

Rap1 is a Ras-related GTPase that is principally involved in integrin- and E-cadherin-mediated adhesion. Rap1 is transiently activated in response to many incoming signals via a large family of guanine nucleotide exchange factors (GEFs). The lack of potent Rap1 dominant-negative mutants has limited the ability to decipher Rap1-dependent pathways. In this study a procedure was developed to generate such mutants consisting in the oligonucleotide-mediated mutagenesis of residues 14-19, selection of mutants presenting an enhanced interaction with Epac2 by yeast two-hybrid screening and counter-screening for mutants still interacting with Rap effectors. In detail analysis of their interaction capacity with various Rap-GEFs in the yeast two-hybrid system revealed that mutants of residues 15 and 16 interacted with Epacs, C3G and CalDAG-GEFI, whereas mutants of position 17 had selectively lost their ability to bind CalDAG-GEFI as well as, for some, C3G. In cellular models where Rap1 is activated via endogenous GEFs, the Rap1[S17A] mutant inhibits both the cAMP-Epac and EGF-C3G pathways, whereas Rap1[G15D] selectively interferes with the latter. Finally, Rap1[S17A] is able to act as a bona fide dominant-negative mutant in vivo since it phenocopies the eye-reducing and lethal effects of D-Rap1 deficiency in Drosophila, effects that are overcome by the overexpression of D-Epac or D-Rap1 (Dupuy, 2005).

The AF-6 homolog Canoe acts as a Rap1 effector during dorsal closure of the Drosophila embryo

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. Canoe (Cno), the Drosophila homolog of mammalian junctional protein AF-6, has been shown to act 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. Rap1 acts upstream of Cno, but 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 (Boettner, 2003).

Rap1 cycles between an inactive GDP-bound and an active GTP-bound state, eliciting distinct downstream responses in the active state. Mammalian Rap proteins were originally identified as antagonists of oncogenic Ras, but more recent studies suggest that the function of Rap1 is largely independent of Ras. 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, in cAMP-induced neurite outgrowth, and, most recently, in the regulation of integrin-mediated cell adhesion and AMPA receptor trafficking during synaptic plasticity (Boettner, 2003 and references therein).

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, 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, 2002). These findings strongly suggest that Rap1 plays a largely Ras-independent role in cell migration and morphogenesis (Boettner, 2003 and references therein).

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. 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 (Boettner, 2003 and references therein).

This study reports 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. 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. cno is required for DC; its protein is localized to the adherens junction and feeds into the JNK pathway by an unknown mechanism. Apart from the fact that it interacts with the ZO-1 homolog Tamou, nothing is known about the regulation of Cno activity at the adherens junction (Boettner, 2003).

Cno has been identified as a protein that interacts with activated Rap1 in a yeast two-hybrid screen. To address the physiological relevance of this interaction, localization studies, a comparative phenotypic analysis, and genetic interaction experiments were undertaken for the two proteins. Rap1 and cno loci are shown to interact synergistically in DC and the physical interaction between Rap1 and Cno is required for DC. The role of Canoe in promoting JNK pathway activity is independent of Rap1 and Canoe therefore has two separate functions in DC (Boettner, 2003).

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. The yeast two-hybrid system was used to identify Drosophila Rap1-specific effector molecules from an embryonic library and several cDNAs encoding Cno were retrieved. Both N-terminal Ras-binding domains (RA1 and RA2) of Cno possess Rap1-binding potential and they interact only with a constitutively active Rap1 mutant, Rap1V12, but not with a dominant negative version of Rap1, Rap1N17, suggesting that Cno may act as an effector for Rap1 (Boettner, 2003).

Several lines of evidence are provided 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, a series of genetic experiments were conducted. They demonstrate that the two molecules act in the same pathway and 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 Rap1N17. Finally, the finding that activated Rap1V12 fails to rescue the cno lof defects indicates that Rap1 acts upstream of Cno. Taken together, the yeast two-hybrid data, colocalization results, and genetic interaction experiments provide comprehensive evidence that Cno functions as a downstream effector of Rap1 in the DC process. These findings represent the first demonstration of a protein acting as a Rap1 effector in vivo (Boettner, 2003).

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 Rap1N17 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 observed in 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 cnoDeltaN 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 Rap1N17 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 Rap1N17 is more severe than that of cno2; ptcGAL4 UAScnoDeltaN). However, no conclusive evidence has been found to support this idea, since the additional effectors of Rap1 identified in the yeast two-hybrid screen have not been investigated for their role in DC (Boettner, 2003).

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. Therefore, use was made of the dominant negative Rap1N17 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 Rap1N17 mutant does not compete well with normal Rap1 for the GEF C3G, calling into question whether this mutant protein can be regarded as a Rap1 dominant negative. But in vivo studies using mammalian Rap1 and now the current study clearly show that Rap1N17 acts as a dominant negative mutant in Rap1 signaling. The successful rescue of Rap1N17 with a concomitantly expressed Rap1wt 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 detected in vitro and the genetic interaction between DRas1 and Cno that influences cone cell formation in the Drosophila eye, have no role during DC (Boettner, 2003).

On which cellular processes might Rap1 and Cno act? 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 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 a study by Knox (2002), where it was 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 the 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. 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. The 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 (Boettner, 2003).

Rap1 GTPase regulation of adherens junction positioning and cell adhesion

Cell-cell junctions are distributed evenly around the lateral circumference of cells within an epithelium. This study found that the even distribution of adherens junctions is an active process that requires the small guanosine triphosphatase Rap1. Cells mutant forRap1 condensed their adherens junctions to one side of the cell. This disrupted normal epithelial cell behavior, and mutant cell clones dispersed into the surrounding wild-type tissue. Rap1 is enriched at adherens junctions, particularly between newly divided sister cells where it may reseal the adherens junction ring. The regulation of adherens junction positioning could play a role in cell mobility and cell division (Knox, 2002).

Cells within an epithelium are linked by several types of junctions. Encircling the apical ends of cells are adherens junctions, which link to the actin cytoskeleton intracellularly and can thereby transmit force across the lateral plane of the epithelium. Although much attention has been paid to the regulation of apico-basal localization of adherens junctions, little is known about the mechanisms that underlie their even distribution around the cell circumference. Rap1 is a small guanosine triphosphatase (GTPase) of the Ras familythat has a role in regulating Drosophila morphogenesis through an undetermined mechanism. During Drosophila wing development, epithelial cells related by lineage normally stay in a coherent group. However, clones of cells mutant for Rap1 dispersed into surrounding wild-type tissue, indicating that loss of Rap1 function disrupts the normal cell-cell adhesion mechanism that keeps lineage-related cells in a coherent group. This phenotype has not been observed for other mutations studied by clonal analysis, including loss-of-function mutations in related GTPases such as Rho1 and Ras85D. Cells lacking Rap1 function still respect the lineage restriction at the anterior-posterior compartment boundary. Observations of shape defects in Rap1 mutant cells suggested that Rap1 might regulate apical cell-cell adhesion. Pupal wing cells mutant for Rap1 lacked the normal hexagonal shape, and the area of the apical, but not the basal, surface was reduced relative to that of wild-type cells. Dispersed mutant cells were often observed in pairs or groups of four cells (Knox, 2002).

To assess the role of Rap1 in cell-cell adhesion, the subcellular localization was examined of adherens junctions and the adjacent, more basal, septate junctions. In contrast to their even distribution around the apical circumference of wild-type epithelial cells, adherens junction components -- including the cell-surface adhesion protein DE-cadherin and two cytoskeletal proteins, α-catenin and β-catenin [visualized with a green fluorescent protein (GFP)] -- were found predominantly on one side ofRap1 mutant pupal wing cells. In a count of 856 cells containing such clusters of adherens junction components, 702 cells (82%) had adherens junctions condensed into a contact with just one neighboring cell. Clusters were also seen between a mutant cell and 2 other mutant cells, or connecting a mutant cell with 3, 4, or 5 neighboring mutant cells. Clusters of adherens junction proteins were observed only at interfaces between mutant cells, and not between a mutant and a wild-type cell. At interfaces between mutant and wild-type cells, normal levels of adherens junctions were observed (Knox, 2002).

Two proteins that may form a molecular link between Rap1 and adherens junctions are the multidomain cytoskeletal linker proteins AF6/canoe and ZO-1. Both AF6 and its Drosophila ortholog canoe bind to activated Rap1, and canoe interacts with ZO-1. Vertebrate ZO-1 binds to the adherens junction component α-catenin, thus completing a possible link from Rap1 to adherens junctions. Both canoe and ZO-1 localize to adherens junctions in normal Drosophila epithelia and like the other adherens junction components, they distributed primarily to one side of Rap1 mutant cells. Although ZO-1 also participates in vertebrate tight junctions and may be present in Drosophila septate junctions, there was not a comparable alteration in septate junction-associated proteins in Rap1 mutant cells. The MAGUK protein Discs large and the band 4.1 ortholog coracle were evenly distributed around the circumference of Rap1 mutant cells. Thus, loss of Rap1 function specifically impairs even distribution of adherens junctions around the cell circumference. The maintenance of septate junctions could explain how Rap1 mutant cells still retain enough cell adhesion to remain within the epithelium (Knox, 2002).

The misplacement of adherens junctions in Rap1 mutant clones suggests that dispersion could be due to sorting caused by differential adhesion. L fibroblasts transfected with P-cadherin sort according to their level of cadherin expression, and such differential adhesion plays a role in Drosophila oocyte positioning at the posterior of the egg chamber. The Rap1 mutant cell-dispersal phenotype may be an additional in vivo example of cell sorting according to differential DE-cadherin-mediated adhesion, although in this case, the amount of adhesion is altered by the failure to distribute adherens junctions evenly around the cell circumference, rather than by altered overall cadherin expression. Provided that the quantity of adherens junction components reflects the strength of adhesion, Rap1 mutant cells could have adhered most strongly to mutant cells on the sides of the cell containing adherens junction clusters, very weakly to other mutant cells, and at normal strength to adjacent wild-type cells. Adhesion between mutant and wild-type cells that was stronger than adhesion between most Rap1 mutant cells could have drawn small groups of mutant cells into wild-type tissue. These results suggest that regulation of the subcellular distribution of cell-cell junctions could play a role in the mobility and invasiveness of cells within an epithelium (Knox, 2002).

Because adherens junctions are also misplaced in undispersed Rap1mutant cells, misplacement is likely to be the cause rather than the consequence of cell dispersal. In this case, mislocalization of adherens junctions during wing development should precede cell dispersal. Clonal cells mutant for Rap1 in the late (wandering) third-instar imaginal disc did not disperse, yet the adherens junction component α-catenin was already mislocalized, indicating that adherens junction mislocalization precedes dispersal. The larvae pupariate within a few hours of this time, and dispersal of Rap1 mutant cells was first observed 2 hours after pupariation. Evagination of the disc during this time period requires extracellular protease activity, which is thought to loosen cell-cell and cell-extracellular matrix contacts, allowing cell rearrangements and shape changes to occur. Cell rearrangements can be observed as the elongation of marked clones; therefore, cells normally exchange neighbors even if they do not normally mix. Loosening of extracellular contacts likely allows Rap1 mutant cells to mix with their wild-type neighbors. Consistent with this, cell dispersion was initially more pronounced at the distal end of the evaginating wing, where cell rearrangements are first initiated (Knox, 2002).

To investigate whether Rap1 recruitment to adherens junctions is involved in aberrant junction distribution in mutant cells, a transgene was expressed encoding a GFP-Rap1 fusion protein. This fusion protein is under the control of the endogenous Rap1 promoter and was expressed ubiquitously throughout development. In normal wing imaginal disc cells, GFP-Rap1 was broadly distributed in the cytoplasm and basolateral membrane and highly concentrated at the position of the adherens junctions, consistent with the possible interaction of Rap1 with adherens junction proteins canoe and ZO-1. Despite its own polarized distribution, Rap1 was not required for normal apico-basal distribution of adherens junctions; α-catenin was located apically in Rap1 mutant imaginal disc clones. β-Catenin and DE-cadherin also did not mislocalize along the apico-basal axis in Rap1 mutant pupal wing clones (Knox, 2002).

The distribution of GFP-Rap1 in dividing cells suggests a mechanism by which Rap1 might normally act to ensure even adherens junction distribution. Dividing cells in the wing imaginal disc retain their adherens junctions with surrounding cells, and the localization of GFP-Rap1 was not altered during division. However, GFP-Rap1 was consistently enriched at the junction between newly formed sister cells. A transient enrichment of GFP-Rap1 between sister cells in the epidermis of living embryos was also observed. Hence, Rap1 may reorganize the adherens junction ring subsequent to or during late cytokinesis to ensure that appropriate amounts of adherens junctions are maintained around the circumference of new cells (Knox, 2002).

One model explaining how loss of Rap1 function during cytokinesis leads to adherens junction clustering is as follows. Maintenance of adherens junction distribution throughout cell division requires a mechanism to convert the single adherens junction ring into two rings, involving breaking and resealing of the ring during cytokinesis. Rap1 could be essential for this process. Failure to reseal the adherens junction ring could allow it to recoil to one side of the cell, driven by contraction of the actin and myosin present in the ring. This would cause rearrangement of cadherin contacts into clusters on sides adjacent to mutant cells with a similar defect, but not on the sides of the cell contacting wild-type cells, where cadherin distribution is stabilized at a normal density. Clusters would most likely form at the interface between sister cells, because both cells' rings recoil at the same time. However, clusters could also form between two adjacent mutant cells that are not sisters if they were in a similar state at the same time. Accordingly, the 14% of clusters between one mutant cell and two others demonstrates that clusters were present at interfaces between cells that are not sisters from their most recent division. Further rounds of division could lead to segregation of clusters into just one daughter cell, producing cells with few adherens junctions, as seen within some Rap1 mutant clones (Knox, 2002).

Rap1 maintains circumferential adherens junction distribution in cells and thus shares with Rho GTPase family members the ability to regulate the cytoskeleton and cell adhesion. Thus, its demonstrated role in morphogenetic processes that are driven by adhesion-dependent cell shape changes and movements may involve regulation of the link between the cytoskeleton and adherens junctions (Knox, 2002).

Drosophila PDZ-GEF, a guanine nucleotide exchange factor for Rap1 GTPase, reveals a novel upstream regulatory mechanism in the mitogen-activated protein kinase signaling pathway

PDZ-GEF is a novel guanine nucleotide exchange factor for Rap1 GTPase. This study isolated Drosophila melanogaster PDZ-GEF (dPDZ-GEF), which contains the all-conserved domains of mammalian and nematode PDZ-GEF including cyclic nucleotide monophosphate-binding, Ras exchange motif, PDZ, RA, and GEF domains. dPDZ-GEF loss-of-function mutants were defective in the development of various organs including eye, wing, and ovary. Many of these phenotypes are strikingly similar to the phenotype of the rolled mutant, implying that dPDZ-GEF functions upstream of the mitogen-activated protein (MAP) kinase pathway. Indeed, it was found that dPDZ-GEF is specifically involved in photoreceptor cell differentiation, facilitating its neuronal fate via activation of the MAP kinase pathway. Rap1 was found to link dPDZ-GEF to the MAP kinase pathway; however, Ras was not involved in the regulation of the MAP kinase pathway by dPDZ-GEF and actually had an inhibitory function. The analyses of ovary development in dPDZ-GEF-deficient mutants also demonstrated another role of dPDZ-GEF independent of the MAP kinase signaling pathway. Collectively, these findings identify dPDZ-GEF as a novel upstream regulator of various morphogenetic pathways and demonstrate the presence of a novel, Ras-independent mechanism for activating the MAP kinase signaling pathway (Lee, 2002).

The Rap1 GTPase functions as a regulator of morphogenesis in vivo

The Ras-related Rap GTPases are highly conserved across diverse species but their normal biological function is not well understood. Initial studies in mammalian cells suggested a role for Rap as a Ras antagonist. More recent experiments indicate functions in calcium- and cAMP-mediated signaling and it has been proposed that protein kinase A-mediated phosphorylation activates Rap in vivo. This study shows that Ras1-mediated signaling pathways in Drosophila are not influenced by Rap1 levels, suggesting that Ras1 and Rap1 function via distinct pathways. Moreover, a mutation that abolishes the putative cAMP-dependent kinase phosphorylation site of Drosophila Rap1 can still rescue the Rap1 mutant phenotype. These experiments show that Rap1 is not needed for cell proliferation and cell-fate specification but demonstrate a critical function for Rap1 in regulating normal morphogenesis in the eye disk, the ovary and the embryo. Rap1 mutations also disrupt cell migrations and cause abnormalities in cell shape. These findings indicate a role for Rap proteins as regulators of morphogenesis in vivo (Asha, 1999).

Activation of the Drosophila C3G leads to cell fate changes and overproliferation during development, mediated by the RAS-MAPK pathway and RAP1

The cellular signal transduction pathways by which C3G, a RAS family guanine nucleotide exchange factor, mediates v-crk transformation are not well understood. This study reports the identification of Drosophila C3G, which, like its human cognate, specifically binds to CRK but not DRK/GRB2 adaptor molecules. During Drosophila development, constitutive membrane binding of C3G, which also occurs during v-crk transformation, results in cell fate changes and overproliferation, mimicking overactivity of the RAS-MAPK pathway. The effects of C3G overactivity can be suppressed by reducing the gene dose of components of the RAS-MAPK pathway and of RAP1. These findings provide the first in vivo evidence that membrane localization of C3G can trigger activation of RAP1 and RAS resulting in the activation of MAPK, one of the hallmarks of v-crk transformation previously thought to be mediated through activation of SOS (Ishimaru, 1999).

Genetic interactions with Rap1 and Ras1 reveal a second function for the fat facets deubiquitinating enzyme in Drosophila eye development

The Drosophila fat facets gene encodes a deubiquitinating enzyme that regulates a cell communication pathway essential very early in eye development, prior to facet assembly, to limit the number of photoreceptor cells in each facet of the compound eye to eight. The Fat facets protein facilitates the production of a signal in cells outside the developing facets that inhibits neural development of particular facet precursor cells. Novel gain-of-function mutations in the Drosophila Rap1 and Ras1 genes are described that interact genetically with fat facets mutations. Analysis of these genetic interactions reveals that Fat facets has an additional function later in eye development involving Rap1 and Ras1 proteins. Moreover, the results suggest that undifferentiated cells outside the facet continue to influence facet assembly later in eye development (Li, 1997).

Biological characterization of Drosophila Rapgap1

The activity of Ras family proteins is modulated in vivo by the function of GTPase activating proteins, which increase their intrinsic rate of GTP hydrolysis. This study has isolated cDNAs encoding a GAP for the Drosophila Rap1 GTPase. Drosophila Rapgap1 encodes an 850-amino acid protein with a central region that displays substantial sequence similarity to human RapGAP. This domain, when expressed in Escherichia coli, potently stimulates Rap1 GTPase activity in vitro. Unlike Rap1, which is ubiquitously expressed, Rapgap1 expression is highly restricted. Rapgap1 is expressed at high levels in the developing photoreceptor cells and in the optic lobe. Rapgap1 mRNA is also localized in the pole plasm in an oskar-dependent manner. Although mutations that completely abolish Rapgap1 function display no obvious phenotypic abnormalities, overexpression of Rapgap1 induces a rough eye phenotype that is exacerbated by reducing Rap1 gene dosage. Thus, Rapgap1 can function as a negative regulator of Rap1-mediated signaling in vivo (Chen, 1997).

Human cDNAs rap1 and rap2 homologous to the Drosophila gene Dras3 encode proteins closely related to ras in the 'effector' region

This study has characterized two new ras-related genes rap1 and rap2 from a human cDNA library, by hybridization with the Drosophila Dras3 gene at low stringency conditions. The rap1 and rap2 genes encode proteins of 184 and 183 amino acid respectively with molecular weights of 20.9 kd and 20.7 kd. These proteins are 53% and 46% identical to the human K-ras protein and share several properties with the classical ras proteins. The C-terminal cysteine involved in the membrane anchoring as well as the GTP binding regions of the p21 ras proteins are present in the rap proteins suggesting that these proteins could bind GTP/GDP and have a membrane localization. The most striking difference between the rap and ras proteins resides in their 61st amino acid. As in the Drosophila Dras3 protein, both rap proteins have a threonine instead of the glutamine found at position 61 of the classical ras proteins. Furthermore the putative effector domain of the ras proteins is strictly conserved in the rap1 protein whereas only one amino acid difference is found in the rap2 protein. This suggests that the rap proteins might interact with the same effector as the ras proteins (Pizon, 1988).

Functions of Rap1 orthologs in other species

The Sema3A receptor Plexin-A1 suppresses supernumerary axons through Rap1 GTPases

The highly conserved Rap1 GTPases (see Drosophila Rap1) perform essential functions during neuronal development. They are required for the polarity of neuronal progenitors and neurons as well as for neuronal migration in the embryonic brain. Neuronal polarization and axon formation depend on the precise temporal and spatial regulation of Rap1 activity by guanine nucleotide exchange factors (GEFs) and GTPases-activating proteins (GAPs). Several Rap1 GEFs have been identified that direct the formation of axons during cortical and hippocampal development in vivo and in cultured neurons. However little is known about the GAPs that limit the activity of Rap1 GTPases during neuronal development. This study investigated the function of Sema3A and Plexin-A1 as a regulator of Rap1 GTPases during the polarization of hippocampal neurons. Sema3A was shown to suppress axon formation when neurons are cultured on a patterned substrate. Plexin-A1 (see Drosophila Plexin) functions as the signal-transducing subunit of receptors for Sema3A and displays GAP activity for Rap1 GTPases. Sema3A and Plexin-A1 suppress the formation of supernumerary axons in cultured neurons, which depends on Rap1 GTPases (Wang, 2018).


Search PubMed for articles about Rap1

Asha, H., de Ruiter, N. D., Wang, M. G. and Hariharan, I. K. (1999). The Rap1 GTPase functions as a regulator of morphogenesis in vivo. EMBO J 18: 605-615. PubMed ID: 9927420

Baril, C., Lefrancois, M., Sahmi, M., Knaevelsrud, H. and Therrien, M. (2014). Apical accumulation of the Sevenless receptor tyrosine kinase during Drosophila eye development is promoted by the small GTPase Rap1. Genetics 197(4): 1237-1250. PubMed ID: 24899161

Boettner, B., Harjes, P., Ishimaru, S., Heke, M., Fan, H. Q., Qin, Y., Van Aelst, L. and Gaul, U. (2003). The AF-6 homolog canoe acts as a Rap1 effector during dorsal closure of the Drosophila embryo. Genetics 165: 159-169. PubMed ID: 14504224

Boettner, B. and Van Aelst, L. (2007). The Rap GTPase activator Drosophila PDZ-GEF regulates cell shape in epithelial migration and morphogenesis. Mol Cell Biol 27: 7966-7980. PubMed ID: 17846121

Bonello, T. T., Perez-Vale, K. Z., Sumigray, K. D. and Peifer, M. (2018). Rap1 acts via multiple mechanisms to position Canoe and adherens junctions and mediate apical-basal polarity establishment. Development 145(2). PubMed ID: 29361565

Borghi, N., Sorokina, M., Shcherbakova, O. G., Weis, W. I., Pruitt, B. L., Nelson, W. J. and Dunn, A. R. (2012). E-cadherin is under constitutive actomyosin-generated tension that is increased at cell-cell contacts upon externally applied stretch. Proc Natl Acad Sci U S A 109: 12568-12573. PubMed ID: 22802638

Bos, J. L. (2005). Linking Rap to cell adhesion. Curr Opin Cell Biol 17: 123-128. PubMed ID: 15780587

Camp, D., Haage, A., Solianova, V., Castle, W. M., Xu, Q. A., Lostchuck, E., Goult, B. T. and Tanentzapf, G. (2018). Direct binding of Talin to Rap1 is required for cell-ECM adhesion in Drosophila. J Cell Sci. PubMed ID: 30446511

Carmena, A., Speicher, S. and Baylies, M. (2006). The PDZ protein Canoe/AF-6 links Ras-MAPK, Notch and Wingless/Wnt signaling pathways by directly interacting with Ras, Notch and Dishevelled. PLoS One 1: e66. PubMed ID: 17183697

Chang, Y. C., Wu, J. W., Hsieh, Y. C., Huang, T. H., Liao, Z. M., Huang, Y. S., Mondo, J. A., Montell, D. and Jang, A. C. (2018). Rap1 negatively regulates the Hippo pathway to polarize directional protrusions in collective cell migration. Cell Rep 22(8): 2160-2175. PubMed ID: 29466741

Chen, F., Barkett, M., Ram, K. T., Quintanilla, A. and Hariharan, I. K. (1997). Biological characterization of Drosophila Rapgap1, a GTPase activating protein for Rap1. Proc Natl Acad Sci U S A 94: 12485-12490. PubMed ID: 9356476

Choi, W., Harris, N. J., Sumigray, K. D. and Peifer, M. (2013). Rap1 and Canoe/afadin are essential for establishment of apical-basal polarity in the Drosophila embryo. Mol Biol Cell 24: 945-963. PubMed ID: 23363604

Ding, J., Tchaicheeyan, O. and Ambrosio, L. (2010). Drosophila Raf's N terminus contains a novel conserved region and can contribute to torso RTK signaling. Genetics 184: 717-729. PubMed ID: 20008569

Dupuy, A. G., L'Hoste, S., Cherfils, J., Camonis, J., Gaudriault, G. and de Gunzburg, J. (2005). Novel Rap1 dominant-negative mutants interfere selectively with C3G and Epac. Oncogene 24: 4509-4520. PubMed ID: 15856025

Ellis, S. J., Goult, B. T., Fairchild, M. J., Harris, N. J., Long, J., Lobo, P., Czerniecki, S., Van Petegem, F., Schock, F., Peifer, M. and Tanentzapf, G. (2013). Talin autoinhibition is required for morphogenesis. Curr Biol 23(18): 1825-1833. PubMed ID: 24012314

Fournier, G., Cabaud, O., Josselin, E., Chaix, A., Adelaide, J., Isnardon, D., Restouin, A., Castellano, R., Dubreuil, P., Chaffanet, M., Birnbaum, D. and Lopez, M. (2011). Loss of AF6/afadin, a marker of poor outcome in breast cancer, induces cell migration, invasiveness and tumor growth. Oncogene 30: 3862-3874. PubMed ID: 21478912

Glading, A., Han, J., Stockton, R. A. and Ginsberg, M. H. (2007). KRIT-1/CCM1 is a Rap1 effector that regulates endothelial cell cell junctions. J Cell Biol 179: 247-254. PubMed ID: 17954608

Goult, B. T., Bouaouina, M., Elliott, P. R., Bate, N., Patel, B., Gingras, A. R., Grossmann, J. G., Roberts, G. C., Calderwood, D. A., Critchley, D. R. and Barsukov, I. L. (2010). Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation. EMBO J 29(6): 1069-1080. PubMed ID: 20150896

Harris, T. J. and Peifer, M. (2004). Adherens junction-dependent and -independent steps in the establishment of epithelial cell polarity in Drosophila. J Cell Biol 167: 135-147. PubMed ID: 15479740

Harris, T. J. and Peifer, M. (2005). The positioning and segregation of apical cues during epithelial polarity establishment in Drosophila. J Cell Biol 170: 813-823. PubMed ID: 16129788

Hoshino, T., Sakisaka, T., Baba, T., Yamada, T., Kimura, T. and Takai, Y. (2005). Regulation of E-cadherin endocytosis by nectin through afadin, Rap1, and p120ctn. J Biol Chem 280: 24095-24103. PubMed ID: 15857834

Huelsmann, S., Hepper, C., Marchese, D., Knoll, C. and Reuter, R. (2006). The PDZ-GEF dizzy regulates cell shape of migrating macrophages via Rap1 and integrins in the Drosophila embryo. Development 133: 2915-2924. PubMed ID: 16818452

Ishimaru, S., Williams, R., Clark, E., Hanafusa, H. and Gaul, U. (1999). Activation of the Drosophila C3G leads to cell fate changes and overproliferation during development, mediated by the RAS-MAPK pathway and RAP1. EMBO J 18: 145-155. PubMed ID: 9878058

Kim, C., Ye, F. and Ginsberg, M. H. (2011). Regulation of integrin activation. Annu Rev Cell Dev Biol 27: 321-345. PubMed ID: 21663444

Knox, A. L. and Brown, N. H. (2002). Rap1 GTPase regulation of adherens junction positioning and cell adhesion. Science 295: 1285-1288. PubMed ID: 11847339

Kooistra, M. R., Dube, N. and Bos, J. L. (2007). Rap1: a key regulator in cell-cell junction formation. J Cell Sci 120: 17-22. PubMed ID: 17182900

Lagarrigue, F., Gingras, A. R., Paul, D. S., Valadez, A. J., Cuevas, M. N., Sun, H., Lopez-Ramirez, M. A., Goult, B. T., Shattil, S. J., Bergmeier, W. and Ginsberg, M. H. (2018). Rap1 binding to the talin 1 F0 domain makes a minimal contribution to murine platelet GPIIb-IIIa activation. Blood Adv 2(18): 2358-2368. PubMed ID: 30242097

Lampugnani, M. G., Orsenigo, F., Rudini, N., Maddaluno, L., Boulday, G., Chapon, F. and Dejana, E. (2010). CCM1 regulates vascular-lumen organization by inducing endothelial polarity. J Cell Sci 123: 1073-1080. PubMed ID: 20332120

Lee, J. H., Cho, K. S., Lee, J., Kim, D., Lee, S. B., Yoo, J., Cha, G. H. and Chung, J. (2002). Drosophila PDZ-GEF, a guanine nucleotide exchange factor for Rap1 GTPase, reveals a novel upstream regulatory mechanism in the mitogen-activated protein kinase signaling pathway. Mol Cell Biol 22: 7658-7666. PubMed ID: 12370312

Li, Q., Hariharan, I. K., Chen, F., Huang, Y. and Fischer, J. A. (1997). Genetic interactions with Rap1 and Ras1 reveal a second function for the fat facets deubiquitinating enzyme in Drosophila eye development. Proc Natl Acad Sci U S A 94: 12515-12520. PubMed ID: 9356481

Linnemann, T., Geyer, M., Jaitner, B. K., Block, C., Kalbitzer, H. R., Wittinghofer, A. and Herrmann, C. (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. PubMed ID: 10224125

Liu, J. J., Stockton, R. A., Gingras, A. R., Ablooglu, A. J., Han, J., Bobkov, A. A. and Ginsberg, M. H. (2011). A mechanism of Rap1-induced stabilization of endothelial cell--cell junctions. Mol Biol Cell 22: 2509-2519. PubMed ID: 21633110

Martin, A. C., Kaschube, M. and Wieschaus, E. F. (2009). Pulsed contractions of an actin-myosin network drive apical constriction. Nature 457: 495-499. PubMed ID: 19029882

McKinley, R. F. and Harris, T. J. (2012). Displacement of basolateral Bazooka/PAR-3 by regulated transport and dispersion during epithelial polarization in Drosophila. Mol Biol Cell 23: 4465-4471. PubMed ID: 23015757

McMahon, A., Supatto, W., Fraser S. E. and Stathopoulos, A. (2008). Dynamic analyses of Drosophila gastrulation provide insights into collective cell migration. Science 322: 1546-1550. PubMed ID: 19056986

McMahon, A., Reeves, G. T., Supatto, W. and Stathopoulos, A. (2010). Mesoderm migration in Drosophila is a multi-step process requiring FGF signaling and integrin activity. Development 137(13): 2167-75. PubMed ID: 20530544

O'Keefe, D. D., Prober, D. A., Moyle, P. S., Rickoll, W. L. and Edgar, B. A. (2007). Egfr/Ras signaling regulates DE-cadherin/Shotgun localization to control vein morphogenesis in the Drosophila wing. Dev Biol 311: 25-39. PubMed ID: 17888420

O'Keefe, D. D., Gonzalez-Nino, E., Burnett, M., Dylla, L., Lambeth, S. M., Licon, E., Amesoli, C., Edgar, B. A. and Curtiss, J. (2009). Rap1 maintains adhesion between cells to affect Egfr signaling and planar cell polarity in Drosophila. Dev Biol 333: 143-160. PubMed ID: 19576205

O'Keefe, D. D., Gonzalez-Nino, E., Edgar, B. A. and Curtiss, J. (2012). Discontinuities in Rap1 activity determine epithelial cell morphology within the developing wing of Drosophila. Dev Biol 369: 223-234. PubMed ID: 22776378

Pizon, V., Chardin, P., Lerosey, I., Olofsson, B. and Tavitian, A. (1988). Human cDNAs rap1 and rap2 homologous to the Drosophila gene Dras3 encode proteins closely related to ras in the 'effector' region. Oncogene 3: 201-204. PubMed ID: 3045729

Plak, K., Pots, H., Van Haastert, P. J. and Kortholt, A. (2016). Direct interaction between TalinB and Rap1 is necessary for adhesion of Dictyostelium cells. BMC Cell Biol 17: 1. PubMed ID: 26744136

Sawyer, J. K., Harris, N. J., Slep, K. C., Gaul, U. and Peifer, M. (2009). The Drosophila afadin homologue Canoe regulates linkage of the actin cytoskeleton to adherens junctions during apical constriction. J. Cell Biol. 186(1): 57-73. PubMed Citation: 19596848

Sawyer, J. K., Choi, W., Jung, K. C., He, L., Harris, N. J. and Peifer, M. (2011). A contractile actomyosin network linked to adherens junctions by Canoe/afadin helps drive convergent extension. Mol Biol Cell 22: 2491-2508. PubMed ID: 21613546

Schmidt, A., Lv, Z. and Grosshans, J. (2018). ELMO and Sponge specify subapical restriction of Canoe and formation of the subapical domain in early Drosophila embryos. Development 145(2). PubMed ID: 29361564

Schwamborn, J. C. and Puschel, A. W. (2004). The sequential activity of the GTPases Rap1B and Cdc42 determines neuronal polarity. Nat Neurosci 7: 923-929. PubMed ID: 15286792

Shirinian, M., Popovic, M., Grabbe, C., Varshney, G., Hugosson, F., Bos, H., Rehmann, H. and Palmer, R. H. (2010). The Rap1 guanine nucleotide exchange factor C3G is required for preservation of larval muscle integrity in Drosophila melanogaster. PLoS One 5: e9403. PubMed ID: 20209136

Siekhaus, D., Haesemeyer, M., Moffitt, O. and Lehmann, R. (2010). RhoL controls invasion and Rap1 localization during immune cell transmigration in Drosophila. Nat Cell Biol 12: 605-610. PubMed ID: 20495554

Spahn, P., Ott, A. and Reuter, R. (2012). The PDZ-GEF protein Dizzy regulates the establishment of adherens junctions required for ventral furrow formation in Drosophila. J Cell Sci 125: 3801-3812. PubMed ID: 22553205

Walther, R. F., Burki, M., Pinal, N., Rogerson, C. and Pichaud, F. (2018). Rap1, canoe and Mbt cooperate with Bazooka to promote zonula adherens assembly in the fly photoreceptor. J Cell Sci 131(6). PubMed ID: 29507112

Wang, H., Singh, S. R., Zheng, Z., Oh, S. W., Chen, X., Edwards, K. and Hou, S. X. (2006). Rap-GEF signaling controls stem cell anchoring to their niche through regulating DE-cadherin-mediated cell adhesion in the Drosophila testis. Dev Cell 10: 117-126. PubMed ID: 16399083

Wang, N., Dhumale, P., Chiang, J. and Puschel, A. W. (2018). The Sema3A receptor Plexin-A1 suppresses supernumerary axons through Rap1 GTPases. Sci Rep 8(1): 15647. PubMed ID: 30353093

Wang, Y. C., Khan, Z. and Wieschaus, E. F. (2013). Distinct Rap1 activity states control the extent of epithelial invagination via α-Catenin. Dev Cell 25: 299-309. PubMed ID: 23623612

Zhu, L., Yang, J., Bromberger, T., Holly, A., Lu, F., Liu, H., Sun, K., Klapproth, S., Hirbawi, J., Byzova, T. V., Plow, E. F., Moser, M. and Qin, J. (2017). Structure of Rap1b bound to talin reveals a pathway for triggering integrin activation. Nat Commun 8(1): 1744. PubMed ID: 29170462

Biological Overview

date revised: 22 February 2022

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