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First published online March 24, 2005
doi: 10.1242/10.1242/dev.01736

Development 132, 1983-1994 (2005)
Published by The Company of Biologists 2005
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The Abelson tyrosine kinase, the Trio GEF and Enabled interact with the Netrin receptor Frazzled in Drosophila

David J. Forsthoefel1, Eric C. Liebl2, Peter A. Kolodziej3,* and Mark A. Seeger1,{dagger}

1 The Ohio State University, Department of Molecular Genetics and Center for Molecular Neurobiology, Columbus, OH 43210, USA
2 Denison University, Department of Biology, Granville, OH 43023, USA
3 Vanderbilt University Medical Center, Department of Cell and Developmental Biology, Center for Molecular Neuroscience, MCN C2210, Nashville, TN 37232-2175, USA



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  		Fig. 1. Abl, trio and ena interact genetically with fra and the Netrin genes. CNS axons were labeled with mAb BP102, and stage 14-16 embryos were dissected and scored for defects. (A) In wild-type embryos, CNS axons are organized into two commissures per segment (AC and PC), and two longitudinal tracts that run the length of the nerve cord on either side of the midline. (B-T) In mutant embryos (genotypes are indicated in the figure), commissure formation is defective. Examples of segments with thin or missing commissures are indicated with arrows in F-H,P and T. Examples of segments with errors in commissural axon pathfinding are indicated with arrowheads in C,H,I,R and S. Examples of breaks in longitudinal pathways are indicated with asterisks in F,H,L,N and O. D,L and N are examples of moderate trio,Abl, fra;Abl, and fra;trio phenotypes, respectively, whereas E,M and O show severe phenotypes. Df(2R)vg135 and Df(3L)FpaI are deficiencies for fra and trio, respectively. Df(1)NP5 removes both NetA and NetB. Anterior is to the top of each image. Scale bar in A: ~25 µm.

 


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  		Fig. 2. Heterozygosity for Abl, trio and ena reduces the severity of inappropriate midline crossing by axons expressing the chimeric Robo-Fra receptor. A subset of longitudinally projecting axons in stage 17 embryos were labeled with mAb 1D4 (anti-Fas2). Examples of Fas2-positive bundles scored as ectopic crossovers are indicated with arrows. (A) In wild-type embryos, Fas2-positive axons project in three distinct longitudinal bundles on either side of the CNS midline, but never cross the midline. (B) In embryos expressing UAS-Robo-Fra under the control of the ELAV-GAL4 postmitotic neuronal driver, numerous Fas2-positive axon bundles cross the midline, reflecting inappropriate attraction towards the midline repellent Slit. (C) In embryos heterozygous for Abl and trio, fewer axon bundles cross the midline. (D) Heterozygosity for Abl and ena also reduces the severity of the Robo-Fra phenotype.

 


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  		Fig. 3. Physical interactions between Fra, Abl and Trio. (A) The cytoplasmic domain of Fra interacts directly with Abl and Trio. Beads only (lane 1), GST bound to beads (lane 2) or GST-FraCYTO bound to beads (lane 3) were incubated with in vitro translated, epitope-tagged Abl or Trio. Approximately 20% of each pulldown (~2 µg of fusion protein) and bound target proteins were resolved by SDS-PAGE. Bound proteins (top two blots) were visualized by anti-Myc immunoblotting, and fusion proteins (bottom gel) were visualized by Coomassie staining. `In' represents 2% of total input protein incubated with beads, GST or GST-FraCYTO. Abl and Trio specifically interact with GST-FraCYTO (lane 3), but not GST or beads (lanes 1 and 2). Trio{Delta}SPR-Myc is deleted for the spectrin-like repeats to optimize expression in vitro. (B,C) Trio and Abl interact directly via their SH3 domains. (B) GST-TrioSH3 (lane 2), but not GST (lane 1), specifically pulls down in vitro translated Abl-Myc. (C) GST-AblSH3 (lane 2), but not GST (lane 1), specifically pulls down in vitro translated Trio{Delta}SPR-Myc. `In' represents ~2.5% of input incubated with GST or fusion protein. Target proteins were visualized by anti-Myc staining, whereas fusion proteins (bottom gel) were visualized by anti-GST staining. Panels B and C were assembled from different lanes on the same gel. (D,E) Fra complexes via its cytoplasmic domain with Abl and Trio in S2 cells. (D) HA-tagged Fra (arrow, lanes 1 and 2, bottom gel) or Fra{Delta}CYTO, a Fra molecule lacking the intracellular domain (arrowhead, lane 3, bottom gel), were co-expressed in S2 cells with Abl-Myc (lanes 2 and 3, middle gel), and complexes were immunoprecipitated with anti-Myc antibody. Fra-HA (arrow, top gel) co-immunoprecipitates only in the presence of Abl-Myc (compare lanes 1 and 2). Fra{Delta}CYTO-HA (arrowhead indicates the absence of Fra{Delta}CYTO-HA, top gel) does not co-immunoprecipitate with Abl-Myc (compare lanes 2 and 3). When expressed in S2 cells, Abl-Myc runs as a ~180-190 kDa doublet, similar to untagged Abl (D, compare with Fig. 4C,D). Additional low-mobility bands in D (top gel, lanes 2 and 3) are background staining of Abl-Myc, which is nearly the same size as Fra-HA. (E) Fra-HA (lane 2) but not Fra{Delta}CYTO-HA (lane 3) co-immunoprecipitates with Trio-Myc.

 


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  		Fig. 4. Trio and Fra are tyrosine phosphorylated in S2 cells. (A,B) Pervanadate treatment results in robust elevation of phosphotyrosine levels in both Trio and Fra proteins. S2 cells transiently expressing Trio-Myc (A) or Fra-Myc (B) were mock-treated with PBS (lane 1) or treated with pervanadate (lane 2) for 30 minutes. Target proteins were immunoprecipitated, and equivalent aliquots of immune complexes were resolved by SDS-PAGE. Blots were probed with either anti-phosphotyrosine (top gel) or anti-Myc (bottom gel). (C,D) Tyrosine phosphorylation of both Trio (C, lane 2) and Fra (D, lanes 2 and 3) is elevated in the presence of increased Abl levels (top gel). S2 cells were co-transfected with 5 µg of pMET Trio-Myc (C) or pMET Fra-Myc (D) and 0, 2 or 5 µg of pMET Abl, as indicated. Twenty-four hours after induction, target proteins were immunoprecipitated and resolved via SDS-PAGE. Blots were probed with anti-phosphotyrosine (top gel), then stripped and re-probed with anti-Myc to verify equivalent loading of samples (middle gel). Approximately 2% of total lysates used in each IP were resolved separately and elevated Abl levels were verified with anti-Abl (bottom gel).

 

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