A role for Tumbleweed/RacGAP50C, and potentially Pav, in postmitotic neurons would predict that these proteins should be expressed there. No endogenous Tum or Pav was detected in the intact CNS by using existing or newly generated antibodies. Therefore epitope-tagged transgenes of Tum and Pav, alone and in combination, were expressed to examine their subcellular localization in MB neurons. A series of GFP-tagged Pav WT (GFP::PavWT) and mutant transgenes have previously been used to study their localization in Drosophila egg chambers and live embryos (Minestrini, 2002 and 2003). It was found that GFP::PavWT not only rescues pav mutants but has similar localization to endogenous Pav, the ring canals (remnants of incomplete cytokinesis rings), and the oocyte nuclei (Minestrini, 2002). When expressed in MB neurons, GFP::PavWT is detected in the nuclei and dendrites and is highly concentrated in the axons of the MB neurons (Goldstein, 2005).
Surprisingly, UAS-MYC::TumWT is enriched in the nuclei when overexpressed in MB neurons. Expression of TumWT tagged with yellow fluorescent protein at its carboxyl terminus revealed a similar nuclear localization, indicating that the nuclear localization is not an artifact of its epitope tag. MYC::TumWT localization was always predominantly nuclear in MB neurons examined in larval and pupal stages (Goldstein, 2005).
Coexpression of UAS-GFP::PavWT and UAS-MYC::Tum alters the subcellular localization of both proteins. These proteins are colocalized in MB neurons, and their localization pattern is a combination of the subcellular distributions of MYC::Tum alone and GFP::PavWT alone. GFP::PavWT is more concentrated in nuclei and less concentrated in axons compared with its expression alone; however, MYC::Tum is now weakly detectable in axons. These observations are consistent with the notion that Tum and Pav bind to each other and regulate each other's subcellular localization in postmitotic neurons (Goldstein, 2005).
Expression of UAS-GFP::PavWT alone or UAS-MYC::TumWT alone does not affect the gross MB axon projection as judged by coexpression with mCD8::GFP. Expression of both UAS-MYC::TumWT and UAS-GFP::PavWT leads to a high occurrence of axon misguidance (14 of 28 MBs examined). The majority of FasII-positive axons form a ball-like structure at their initial trajectory, and only a small subset of axons is able to extend beyond this ball-like structure into the medial lobe. In other cases, axons fail to enter their correct path at the branch point for the dorsal and medial lobe. One possible explanation is that cytoplasmic accumulation of Tum, by means of Pav binding, disrupts axon development (Goldstein, 2005).
To further test this hypothesis, use was made of a Pav mutant transgene. Mutations in three of the nuclear localization signals of Pav (PavNLS) blocks the nuclear localization of Pav protein. As predicted, the nuclear localization of GFP::PavWT, but not its dendritic or axonal localization, is disrupted in GFP::PavNLS. GFP::PavNLS does not lead to any gross morphological changes when expressed alone. As with UAS-GFP::PavWT, coexpression of GFP::PavNLS and MYC::Tum results in altered subcellular localization of both proteins: GFP::PavNLS is localized to the nucleus, and more MYC::Tum is 'dragged' out of the nucleus. Interestingly, almost all MB axons are misrouted under this condition. In addition to forming a cluster of axons in a ball-like form as with GFP::PavWT, axons bypass their normal path via the peduncle and instead project medially toward the front of the brain just below the normal medial lobe and then project dorsally to innervate the medial lobe. The gain-of-function phenotype seen with coexpression of Tum and Pav suggests that they form a complex that disrupts the normal development of axons. The occurrence and severity of this axon misrouting is increased when the Pav transgene has a decreased drive to enter the nucleus, indicating that Pav may act as a transporter of Tum and that local concentrations of Tum in the axon directly influences its capacity to affect axonal development (Goldstein, 2005).
To test whether the GAP activity is necessary for the axon misrouting, a point mutation (TumR417L) of the conserved arginine residue critical for GAP activity was introduced into the UAS construct. As with the WT form, UAS-MYC::TumR417L localizes to the nuclei of MB neurons when expressed in MB neurons and does not lead to morphological phenotypes. When coexpressed with UAS-GFP::PavNLS, the subcellular localization of MYC::TumR417L and GFP::PavNLS reflects a pattern similar to that seen with WT Tum coexpression, with MYC::TumR417L detected in both axons and in the nuclei of MB neurons. However, with the loss of the conserved arginine in the GAP domain, axon disruption capability of Tum is almost completely abolished, suggesting that the GAP activity of Tum mediates the gain-of-function phenotype seen with Tum and Pav coexpression (Goldstein, 2005).
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