Frazzled and Abelson as only 21% of embryos still show a minor crossover or two ([Figure 2E]; quantified in [Figure 2G]). Confirming the BP102 results, these crossing defects depend on the presence of Fra protein as expressing Frawt with the 1407-Gal4 driver line restores the crossover defects [Figure 2G, H]. Expression of FraDP1 or FraDP2 also restores these crossing errors similar to Frawt [Figure 2G]. However, expression of FraDP3 significantly (P,0.01) reduced the expressivity and penetrance of the crossing errors [Figure 2G, 2I]. Thus, the interaction between Fra and either Ablwt or BcrAbl during commissure formation does not depend on a specific P-motif. On the other hand, BcrAbl selectively interacts with the P3 motif of Fra to induce ectopic midline crossing errors. This is consistent with the reduction in fuzzy commissures noted with BP102 [Figure 1]. Abl and BcrAbl bind to Fra independent of a specific P- motif Our genetic rescue data suggest that Fra regulates Abl activity independent of a specific P-motif, while the P3 motif may have a special interaction with hyperactivated BcrAbl. To further address this, we asked whether these genetic interactions are reflected in a physical interaction between Abl and Fra. Forsthoefel et al. [36] noted that Abelson binds to the cytoplasmic tail of Frazzled but did not determine if the binding required a specific P-motif. Using Drosophila S2 cells, we created two sets of stable lines expressing either HA- or myc-tagged wild-type or P- motif deletion mutants of Fra under the control of a metallothio- nine promoter. When compared to the level of HA-tagged receptor pulled down in these immunoprecipitation assays, endogenous Abl continues to bind to Frazzled even when an individual P-motif (DP1, DP2 and DP3) is deleted [Figure 3A]. Similar results were observed using the myc-tagged versions of wild-type Fra and its P-motif deletions. When co-expressed in S2 cells, both wild-type (Ablwt) and kinase inactive (AblKN) transgenes bind to wild-type Fra as long as the cytoplasmic domain is present [Figure 3B], although it is noted that only a small fraction of the available protein is found in complex with each other. Figure 3. Both Ablwt and BcrAbl bind to Frazzled independently of a specific P-motif. [A] Stable Drosophila S2 cells were created that express an HA-tagged form of wild-type [wt] or P-motif deletion (DP1, DP2, DP3) mutants of Frazzled (Frax-HA). Frazzled was immunoprecipitated (IP) from cells using a mouse monoclonal anti-HA, and western blots were probed (IB) with guinea pig anti-Drosophila Abl (D-Abl; top) or rabbit anti-HA (bottom). Compared to the amount of Frazzled receptor pulled down, endogenous Abl binds to Frazzled independently of a P-motif. [B] Transiently transfected Ablwt or AblKN (kinase inactive) genes also bind to a myc-tagged version of wild-type Fra (Frawt-myc])but not to a HA-tagged receptor missing its cytoplasmic domain (FraDC-HA). The amount of Fra (middle row) and Abl (bottom row) present in the lysates is indicated. [C] Activated BcrAbl, detected by a rabbit antibody against the Bcr region, also binds to Fra-myc even if one of the P-motifs is removed. No band is observed in mock IPs from S2 cells expressing only BcrAbl (far left). The amount of Fra pulled down (middle row), and the amount of BcrAbl available in lysates (bottom row) is shown. [D] In the top panel, S2 cells were pretreated for 30 min with pervanadate and then wild-type or P-motif deletions of Frazzled were immunoprecipitated using mouse anti-myc and the degree of tyrosine phosphorylation assessed by probing a blot with mouse anti- phosphotyrosine (a-pY; top row). Compared to the amount of receptor pulled down (bottom row), the level of tyrosine phosphorylation does not appear to be diminished when a P-motif is deleted. The bottom panel assesses Frawt phosphorylation when Abl transgenes are co-expressed in S2 cells that were not pretreated with pervanadate. Compared to the level observed in S2 expressing only Frawt-myc, phosphotyrosine levels increase slightly if cells are co-transfected with Ablwt or BcrAbl, but not AblKN (bottom blot). In this blot, autophosphorylation of BcrAbl denoting its activated state is apparent (arrowhead). The position of molecular weight standards (in kDa) is indicated on the left side of each panel. doi:10.1371/journal.pone.0009822.g003 PLoS ONE | www.plosone.org 6 March 2010 | Volume 5 | Issue 3 | e9822