Individual P-motifs were deleted as defined in Kolodziej et al. [13]. pMT-FraDP1 is deleted for PPDLWIHHDQMELKNIDK; pMT- FraDP2 is deleted for TIESSKRGHPLKSFSVPGPPPTG- GATPVTKHTP; pMT-FraDP3 is deleted for ELNQEMAN- LEGLMKDLSAITANEFEC. All three P-motif deletion con- structs contain a C-terminal HA epitope tag. To make myc- tagged versions, we first modified a commercially available S2 cell metallothionine vector (pMT-V5His, Invitrogen) to include a hygromycin resistance cassette (herein pMT-Hygro), and then introduced a myc epitope tag and tobacco etch virus protease site (TEV) just before this vector’s V5 epitope and His tags. Then, using the HA-tagged versions as templates, we PCR amplified Fra wild-type (Frawt) and P-motif deletions (FraDP1, FraDP2, FraDP3) and cloned them into unique SpeI and PvuI sites of this parental vector. S2 cell culture and Immunoprecipitation Studies Drosophila S2 cells were maintained in Schneider’s media supplemented with 10% fetal bovine serum (complete media) at 27uC as per the conditions in Drosophila Expression System (Invitrogen). Approximately 26106 cells were transfected (Effectene, Qiagen) with either myc- or HA-tagged FraWT, FraDP1, FraDP2, and FraDP3 using the procedure recommended by the manufacturer. For the HA-tagged version, selection was achieved by co-transfection with pCohygro. Stable lines were selected over several weeks using 300 mg/ml hygromycin (Invitrogen). Fra protein expression was induced with 700 mM copper sulfate, and, 24 hours later, Fra was activated by adding a mixture of Netrin A and Netrin B for 30 minutes at 27uC. The cells were harvested by centrifugation at 13000 rpm and lysed in NP-40 buffer [36]. Cell extracts were pre-cleared by incubating with goat anti-mouse IgG magnetic beads (Pierce) for 30 minutes at 4uC. Following a two-hour incubation with 1 mg mouse anti-HA antibody (Sigma), Frazzled-antibody complex was pulled down using the magnetic IgG beads. The bead- protein complex was boiled in 1X SDS sample buffer and loaded onto a 7.5% poly-acrylamide gel. Protein was transferred to PVDF membrane (Millipore) following standard procedures and probed with antibody. Fra pull down was confirmed by blotting against the epitope tag using rabbit anti-HA or anti- myc (Sigma) at 1:10000. After stripping (50 mM Tris (pH 6.8), 2% SDS and 100 mM b-mercaptoethanol at 50uC for 30 minutes), membrane was re-probed with either rabbit anti- Drosophila Abl (kindly provided by David VanVactor), guinea pig anti-Drosophila Abl (kindly provided by Mark Peifer) or rabbit anti-Bcr (Cell Signaling) at 1:1000 dilution. The amount of Fra pull down was confirmed using a rabbit anti-myc or HA antibody (Sigma). For co-transfections with Abl transgenes, stable lines expressing full length Fra or mutants were transiently co-transfected with pMT GAL4 and either UAS- Ablwt, UAS-AblKN, or UAS-BcrAbl (1:20). Twenty-four hours later, the cells were induced for protein expression and complexes were isolated as described above. To examine tyrosine phosphorylation of Fra and its P-motif deletions, the above procedure was used except that Frazzled expressing cells were treated with pervanadate [36] before Netrin was added. Phosphotyrosine was detected using PY-20 (Upstate) at 1:1500 and 1:20000 goat anti-mouse-HRP (Jackson). Results Previous studies showed that loss-of-function mutations in abl enhance the degree of commissure loss in a fra mutant [36] while over-expression of a constitutively active form of Abl (Bcr210Abl, Frazzled and Abelson herein just BcrAbl) induces axons to ectopically cross the midline especially when wild-type Fra (Frawt) is co-expressed [37]. Thus, it seems possible that Abl helps mediate midline attractive signaling, and if so, a simple prediction would be that over- expressing wild-type Abl, or BcrAbl in a fra homozygote would partially rescue fra-dependent defects in commissure formation. Accordingly, using the GAL4–UAS system and previously characterized stocks [32,37], we over-expressed UAS-Ablwt (U- Ablwt) or UAS-BcrAbl (U-BcrAbl) in all neurons of a homozy- gous fra mutant (fra3/fra4) embryo and assessed commissure formation using the monoclonal antibody BP102. This antibody stains the anterior and posterior commissures (AC and PC, respectively) as well as longitudinal connectives (LC) running on either side of the midline [Figure 1A]. We were surprised to find that, rather than rescuing fra3/fra4 commissural defects, elevating Abl activity actually enhanced the loss of commissures ([Figure 1E, F] black arrowhead, PC; white arrowhead, AC). Another striking defect was also observed: large bundles of axons exiting the CNS, often extending beyond the CNS/PNS boundary ([Figure 1E, F] arrows) (herein designated AEP, Axons Exiting to Periphery). Fra interacts with Abl to form commissures One of the predominant defects in fra3/fra4 mutants is thinning and missing posterior commissures, with nearly a quarter [24%] of segments showing this defect ([Figure 1D], black arrowhead; graphed in K; refs [13,32,36]). Over- expression of BcrAbl significantly (P,0.001) enhances these defects nearly three fold with 76% of segments having a thin or missing PC ([Figure 1E], black arrowhead; quantified in K). In addition, the AC, which is virtually unaffected in fra3/fra4 homozygote, was thin or missing in 41% of segments (P,0.01; [Figure 1E], white arrowhead; graphed in K). This reduction in commissural axons crossing the midline was particularly surprising since expression of BcrAbl in a wild-type embryo results in fuzzy commissures, a defect caused by extra axons crossing the midline (see [37]). Fuzzy commissures remain evident in a fra3 heterozygote ([Figure 1B], black arrowhead; see below). To test whether this result was unique to BcrAbl, we expressed wild-type Abl (U-Ablwt) in a fra3/fra4 mutant background. In wild-type embryos, expression of Ablwt alone is innocuous [49], although a rare commissure may be malformed in a fra3 heterozygote (see arrowhead [Figure 1C]). When Ablwt is expressed in a fra3/fra4 mutant the phenotype is strikingly similar to that caused by expression of BcrAbl, with a three-fold (P,0.01) enhancement in PC defects and the emergence of AC defects ([Figure 1F], note that AEP defects are also present, black arrow; quantified in K). The enhancement in commissure defects partially requires Abl kinase activity. While pan-neural expression of a kinase dead version of Abl (U-AblKN) on its own has no phenotype in either wild-type or fra heterozygous embryos, expression in a fra3/fra4 mutant still significantly (P,0.05) increases the frequency of thin and missing PC defects to about half that observed with over-expression of either Ablwt or BcrAbl; expression of AblKN spares the AC [Figure 1K]. Thus, both increasing and decreasing [36] Abl activity in the absence of Fra reduces the ability of commissural axons to cross the midline. It is intriguing that such a striking phenotype is uncovered when Fra is removed since pan-neural expression of Ablwt on its own has very few defects and pan-neural expression of BcrAbl actually causes axons to erroneously cross the midline [49,57]. This is also true when Abl signaling is increased in PLoS ONE | www.plosone.org 3 March 2010 | Volume 5 | Issue 3 | e9822