BIOLOGICAL OVERVIEW

Rho family small GTPases, including Rho, Rac, and Cdc42, are strong candidates for transducing axon guidance information to the actin cytoskeleton. In nonneuronal cells, these molecular switches control distinct types of actin-based cell motility in response to extracellular cues. Work in nonneuronal systems indicates that small GTPase activation is initiated by the Dbl homology (DH) family of guanine nucleotide exchange factors (GEFs), which allow Rho family GTPases to release GDP and acquire the GTP necessary for the active state. It is thought that association of GEF proteins with complexes that bind the cytoplasmic domains of cell surface receptors provides the necessary link between extracellular cues and GTPase activation. Described here is the Drosophila homolog of Trio, a multidomain protein containing two DH domains whose vertebrate counterpart specifically activates Rac and Rho (Debant, 1996). A close relative of Trio has also been identified in C. elegans (unc-73; Steven, 1998), in which mutant analysis reveals a variety of axon guidance and cell migration phenotypes (Siddiqui, 1991; McIntire, 1992). Drosophila trio mediates the development of multiple embryonic axon pathways. trio shows dosage-sensitive, reciprocal genetic interactions with Abl tyrosine kinase, revealing Trio's role in axon pathfinding (Liebl, 2000). Thus genetic analysis in Drosophila reveals potent genetic interactions between trio and a number of signaling components thought to control actin dynamics, including Rac, Dlar, and components in the Abl tyrosine kinase pathway (Bateman, 2000). Trio is distributed along axons in the central nervous system (CNS) of embryos and is strongly expressed in subsets of brain regions, including the mushroom body (MB). Loss-of-function trio mutations result in the misdirection or stall of axons in embryos and also cause malformation of the MB. The MB phenotypes are attributed to alteration in the intrinsic nature of neurites, as revealed by clonal analyses. Thus, Trio is essential in order for neurites to faithfully extend on the correct pathways (Bateman, 2000; Liebl, 2000; Awasaki, 2000).

Cell transfection and in vitro nucleotide exchange assays with each DH domain of human Trio have suggested that GEF1 preferentially activates Rac, whereas GEF2 activates Rho (Debant, 1996; Bellanger, 1998a). To explore the relationship between trio and Rac in Drosophila, the compound eye was used as an established system to test for genetic interactions in GTPase signaling pathways. Rac overexpression under the control of the eye-specific promoter GMR creates a mispatterened 'rough' eye in which individual ommatidia are misshapen. However, removal of a single copy of trio causes a dramatic suppression of this Rac gain-of-function phenotype. This is true for additional trio alleles. In contrast, overexpression of Rho also generates a rough eye, but this phenotype is not significantly altered by reduction in trio activity. This suggests that in the Drosophila retina, trio functions to activate one or more of the Drosophila Rac-like genes but not Rho (Bateman, 2000).

In the embryo, previous studies have shown that the same nerve branches affected by trio mutations are also most sensitive to Rac perturbation. Although occasional ISNb stop short phenotypes are observed, the predominant ISNb and SNa bypass phenotypes induced by Drac1^N17 overexpression are distinct from phenotypes caused by loss of trio function. This difference likely reflects Drac1^N17 interference with multiple neural activators of Rac GTPases. However, it was reasoned that if trio is involved in Rac activation in the embryonic motor nervous system, the penetrance of the Drac1^N17 phenotype should be sensitive to changes in the genetic dose of trio. Consistent with this hypothesis, removal of a single copy of trio in embryos expressing Drac1^N17 causes a distinct increase in the penetrance of ISNb bypass. This was true for all alleles of trio tested. Moreover, coexpression of Drac1^N17 and a wild-type trio transgene results in a dramatic suppression of the ISNb bypass phenotype, consistent with the model that trio is an activator of Rac GTPases in the embryonic motor nervous system (Bateman, 2000).

Despite phenotypic differences between Drac1^N17 and trio in the motor nervous system, analysis of the CNS in embryos lacking Rac function reveals defects identical to those observed in trio mutants. Specifically, expression of the dominant-negative Drac1^N17, under the control of the neural-specific GAL4 driver C155, causes a failure of the lateralmost Fas II-positive longitudinal pathway to properly connect at stage 17 (13.7%). In contrast, neural expression of either Dcdc42^N17 or DRho1^N19 does not cause defects in longitudinal pathfinding, indicating that the CNS phenotype is specific to interference with Rac-like GTPase function. Thus, in the CNS, mutant phenotypes of Drac1^N17 and trio are consistent with disruption of a common pathway (Bateman, 2000).

The ISNb and longitudinal pathway defects observed in trio mutants are similar to those of phenotypes observed in embryos mutant for the Abl tyrosine kinase. Previous analysis has shown that a partial reduction in Abl function suppresses the bypass phenotype caused by mutations in the RPTP Dlar, implying an antagonistic relationship between kinase and phosphatase. To address trio function at this ISNb choice point, ISNb pathfinding was examined for dosage-sensitive interactions between Dlar and trio. In strong zygotic Dlar mutants, ISNb bypass was observed at a moderate frequency (18.4%, A2-A7 hemisegments). However, partial reduction of trio activity in this Dlar background enhances the ISNb bypass ~2-fold. Although this potentiation disagrees with a simple model in which trio and Abl function together to oppose phosphatase signaling, it is consistent with the observation that neural expression of Drac1^N17 enhances the frequency of bypass in Dlar mutants. Thus, although trio may collaborate with Abl at the CNS midline, it rather appears to cooperate with Dlar and Drac1 during ISNb ventral target entry. The absence of bypass phenotypes in trio single mutants is likely to reflect the existence of additional inputs to Rac family GTPases that would be susceptible to the Drac1^N17 dominant-negative effect (Bateman, 2000).

While the functional connection between Trio and Rac-like GTPases provides clear links to cytoskeletal events, recent results suggest that Trio may coordinate the activities of multiple signaling partners. In addition to genetic interactions between Drosophila trio, Abl, ena, and Dlar, vertebrate Trio has been shown to bind directly to Filamin (Bellanger, 1998b), a protein required for cell motility in a variety of cell types. In addition to forming direct orthogonal cross-links between microfilaments, characteristic of lamellipodia, Filamin also plays a role in RalA GTPase-mediated induction of filopodial structures and interacts with cell surface receptors. Recent data also reveal a role for Drosophila filamin in the construction of actin-based structures during oogenesis (Li, 1999; Sokol, 1999) and in the formation of embryonic axon pathways (N. Sheard, J. B., T. Hays, and D. V. V., unpublished data cited in Bateman, 2000). Thus, Filamin may provide another direct link between Trio and the cytoskeletal arrays that drive the motile leading edge (Bateman, 2000).

Additional partners for Trio may also come from genetic studies. For example, previous genetic screens reveal another gene with a trio-like ISNb phenotype called 'stop short' (or 'shot', also known as 'kakapo'); recent molecular cloning reveals that this gene encodes a protein with an actin-binding domain. Interestingly, kakapo is expressed in the same muscle attachment sites in which trio is expressed, suggesting a functional overlap in different cell types. The shared function of cytoskeletal proteins in many different morphogenetic events is a common theme in many organisms. While these important proteins and signaling pathways are used again and again, their roles in a particular context may be quite specific. The challenge for the future is to dissect the specific from the general in order to understand the information content of the many pathways that regulate cytoskeletal structure and dynamics (Bateman, 2000 and references therein).

Recent genetic and biochemical analyses have suggested functional interactions between tyrosine phosphorylation and microfilament dynamics in the growth cone. In Drosophila second site modifier screens have found that heterozygosity for enabled (ena) alleviates the Abl mutant phenotype. Enabled can bind to and be phosphorylated by Abl and can bind to and be dephosphorylated by Dlar; Enabled's phosphorylation state is believed to influence its protein-protein interactions. Enabled can bind Profilin and is colocalized with Abl and Dlar in axons. Tyrosine phosphorylation of Enabled may be one way to influence F-actin production in the growth cone and therefore may be an important influence on Drosophila axonal pathfinding. Work in mice suggests that similar molecular cascades function in mammals; Mena, which promotes actin polymerization and binds Profilin, is concentrated in filopodia tips of growth cones, and Mena null mice have axonal projection defects (Liebl, 2000 and references therein).

Genetic analysis in Drosophila has been used to identify a variety of other genes having dosage-sensitive interactions with Abl, since dosage-sensitive genetic interactions can occur between members of signal transduction networks. Reducing the gene dose by half of either disabled (dab), failed axon connections (fax), or Notch has been shown to worsen the Abl mutant phenotype, while reducing the dose of Abl by half has been shown to worsen the profilin (chic) mutant phenotype. Reducing the gene dose of Abl has also been shown to alleviate the Dlar mutant phenotype. It is likely that these genes' products are integrated into molecular networks regulating growth cone guidance, although the biochemical interaction between these molecules and Abl is not yet established in all cases (Liebl, 2000 and references therein).

A dosage-sensitive genetic interaction has been shown to occur between Abl and Trio. Not only do heterozygous mutations in trio dramatically enhance the Abl mutant phenotype, but reducing the gene dose of either Abl, fax, or dab enhances the trio mutant phenotype. Heterozygosity for ena partially alleviates the trio mutant phenotype. These dosage-sensitive genetic interactions between trio, Abl, and genes believed to be in Abl-mediated signal transduction networks, as well as the potential for Trio to regulate the actin cytoskeleton by influencing small GTPases, position the Trio protein as another candidate for the integration of tyrosine phosphorylation and microfilament dynamics in the growth cone (Liebl, 2000).

GENE STRUCTURE

cDNA clone length - 8033 bp

Bases in 5' UTR - 2099

Bases in 3' UTR - 347

PROTEIN STRUCTURE

Amino Acids - 2264

Structural Domains

trio was isolated by PCR based on its proposed similarity to mammalian Trio proteins (Bateman, 2000). Two other studies have identified trio on the basis of either a mutational phenotype (Liebl, 2000) or on the basis of sequence homology revealed in database searches (Awasaki, 2000). The predicted Trio peptide sequence was compared to the sequence of hTrio, its close relative, Kalirin (Alam, 1997), and UNC-73, as well as other less related proteins. Analysis of the Drosophila sequence using BLAST and PFAM algorithms predicts the presence of seven N-terminal spectrin-like domains (residues 313-1227) followed by a DH domain (DH1, residues 1287-1486), a pleckstrin homology domain (PH1, residues 1493-1581), a Src-homology 3 domain (SH3, residues 1645-1708), a second DH domain (DH2, residues 1945-2122), and a second PH domain (PH2, residues 2136-2245). Both DH domains contain all of the highly conserved residues in the GTPase-binding surface shown to be functionally important by site-directed mutagenesis (Liu, 1998) and are thus likely to be enzymatically functional (Bateman, 2000).

The relative amino acid identity for each domain and the organization of these domains reveal that the Drosophila sequence is not only conserved in overall structure with members of the Trio GEF family, but is in fact most closely related to hTrio. Despite the close relationship between these family members in fly, human, and worm, all proteins of this family are divergent in their extreme C-terminal domains. Although Drosophila Trio contains only 15 residues C-teminal to the PH2 domain, hTrio contains a serine/threonine kinase domain in this position (Debant, 1996), whereas UNC-73 lacks the kinase and instead contains a fibronectin-like domain (Steven, 1998). The functional significance of this diversity is currently unknown (Bateman, 2000). Although human Trio contains a C-terminal Ig-like domain, a second SH3-like domain, and a serine protein kinase domain (all of which are lacking in Drosophila Trio), the overall arrangement of the N-terminal domain, the spectrin repeats, the DH/PH domains, and the first SH3-like domain is highly similar in both Drosophila Trio and human Trio (Liebl, 2000).

date revised: 4 August 2000

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