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doi: 10.1242/10.1242/dev.00545

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Interactions between the secreted protein Amalgam, its transmembrane receptor Neurotactin and the Abelson tyrosine kinase affect axon pathfinding

Eric C. Liebl^1,, R. Grant Rowe¹, David J. Forsthoefel², Amanda L. Stammler¹, Erica R. Schmidt^1,*, Michelle Turski^2, and Mark A. Seeger²

¹ Department of Biology, Denison University, Granville, OH 43023, USA
² Department of Molecular Genetics and The Center for Molecular Neurobiology, The Ohio State University, Columbus, OH 43210, USA
^* Present address: College of Medicine, The Ohio State University, Columbus, OH 43210, USA
Present address: University Program in Genetics, Duke University, Durham, NC 27708, USA

View larger version (35K): [in a new window]   Fig. 2. Biochemical characterization of Ama+ and AmaM109. (A) The organization of Ama with its signal sequence and three Ig domains (loops). The N terminus is towards the left. The amaM109 and the amaR1 mutations are also indicated. (B) Representative photomicrographs showing our S2 cell adhesion assay at t=8 hours. In the top three panels, all cells were engineered to express wild-type Nrt and were exposed to either control (left), Ama+-containing (center) or AmaM109-containing (right) conditioned media. In the bottom two panels, cells were engineered to express membrane-anchored forms of Ama. (C) Quantification of our S2 cell adhesion assays. Bars represent the percent of total particles at t=0 hours counted at t=8 hours. Values represent the average of two independent experiments; error bars show the standard error of the mean. (D) Anti-Ama immunoblots. The arrow indicates the mobility of full-length Ama. Lanes 1, 2: equal amounts of Ama+-conditioned media or of AmaM109-conditioned media were resolved. These lanes show that comparable amounts of Ama were present in both types of conditioned media. Lanes 3-6: immunoblots of S2 cell lysates from cell pull-down assays. Equivalent amounts of cell lysates were loaded in each lane. Lane 3: naïve S2 cells exposed to Ama+-conditioned media. Lane 4: Nrt-expressing S2 cells exposed to Ama+-conditioned media. Lane 5: naïve S2 cells exposed to AmaM109-conditioned media. Lane 6: Nrt-expressing S2 cells exposed to AmaM109-conditioned media. These lanes show that Ama+ and AmaM109 bound specifically to S2 cells expressing Nrt. (E) Anti-Nrg immunoblots. Equivalent amounts of cell lysates were loaded in each lane. The arrow indicates the mobility of the Ama-Nrg fusion protein. Lane 1: S2 cells expressing Ama+-TM. Lane 2: S2 cells expressing AmaM109-TM. These lanes show equivalent amounts of these chimeric proteins were expressed.

View larger version (134K): [in a new window]   Fig. 1. CNS phenotypes of Abl and ama mutants. Embryos from stage 14/15 were stained with mAb BP102 and CNSs were dissected. Representative examples of the overall phenotype associated with each genotype are shown. (A) Abl1/Abl4, (B) Abl1, amaM109/Abl4, (C) Abl1, Df(3R)ama/Abl4, (D) Abl1, amaR1/Abl4, (E) Abl1, amaR1/Abl4, Df(3R)ama, (F) Abl1, amaM109/Abl4, Df(3R)ama. Percentages below each panel indicate the percent of segments with commissure defects; n=total segments scored. Arrows in B,E show typical examples of segments scored as having defective commissures.

View larger version (132K): [in a new window]   Fig. 3. CNS phenotypes of Abl and nrt mutants. Mutants were either generated by RNAi (A-C) or by zygotic genotypes (D-F). Embryos from stage 14/15 were stained with mAb BP102, and CNSs were dissected. Representative examples of the overall phenotype associated with each genotype are shown. (A) Abl null, (B) nrt null, (C) Abl and nrt null, (D) Abl1, nrtM54/Abl4, (E) Abl1, nrtM100/Abl4, (F) Df(3L)st-j7, nrtM2/Abl1, nrtM54. Percentages below each panel indicate the percent of segments with commissure defects; n=total segments scored. Arrows in D,E show typical examples of segments scored as having defective commissures.

View larger version (20K): [in a new window]   Fig. 4. Biochemical characterization of NrtM100 and NrtM221. (A) The Nrt protein is represented. The N terminus is towards the left. The location of mutations nrtM29, nrtM221, nrtM54, nrtM2 and nrtM100 are also shown. nrtM54 is an 11 bp deletion that results in a frameshift (fs). (B) Graphical representation of the quantification of our S2 cell adhesion assay. Bars represent the percent of total particles at t=0 hours counted at t=8 hours. Values represent the average of two independent experiments; error bars show the standard error of the mean. (C) Immunoblots of S2 cell lysates from cell pull-down assays. (Top) Anti-Nrt immunoblots. Equivalent amounts of cell lysates were loaded in each lane. The arrow shows the migration of full-length Nrt. Lane 1: naïve S2 cells. Lane 2: S2 cells engineered to express Nrt+. Lane 3: S2 cells engineered to express NrtM100. Lane 4: S2 cells engineered to express NrtM221. Naïve S2 cells did not express Nrt, and all Nrt proteins were expressed from the pMET plasmid at comparable levels. (Bottom) Anti-Ama immunoblots. Equivalent amounts of cell lysates were loaded in each lane. The arrow shows the migration of full-length Ama. Lane 1: naïve S2 cells. Lane 2: Nrt+-expressing S2 cells. Lane 3: NrtM100-expressing S2 cells. Lane 4: NrtM221-expressing S2 cells. Only S2 cells expressing wild-type Nrt bound Ama.

View larger version (10K): [in a new window]   Fig. 5. Cytological region 72D; 73E. The cytological map is represented across the top; the centromere is towards the right. The placements of the fax, Abl, dab and nrt genes are shown. Solid lines represent the cytological extent of the chromosomal deficiencies as reported in FlyBase (http://flybase.bio.indiana.edu). The genotype of these deficiencies with regard to fax, Abl, dab and nrt is indicated to the right. Based on data reported previously (Henkemeyer et al., 1987), deficiencies that dominantly enhance the Abl mutant phenotype are indicated by asterisks. Although the distal breakpoint of Df(3L)st100.62 is listed as 72F3-4 in FlyBase and fax is placed in 72E5-F1, Hill et al. (Hill et al., 1995) report that Df(3L)st100.62 is null for fax. In(3L)std11 contains the deficiency shown as well as an inversion (73E1-2).