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First published online 4 April 2007
doi: 10.1242/dev.02842

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The F-actin-microtubule crosslinker Shot is a platform for Krasavietz-mediated translational regulation of midline axon repulsion

¹ Department of Cell and Developmental Biology, School of Dentistry, Seoul National University, Seoul 110-740, Republic of Korea.
² School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea.
³ Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232-2175, USA.
⁴ Department of Physiology, School of Dentistry, Seoul National University, Seoul 110-740, Republic of Korea.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Shot is physically associated with the translation initiation factor Kra/eIF5C. (A) Domain structure of the evolutionarily conserved Shot isoforms, Shot L(A) and Shot L(B). ABD, actin-binding domain; CH, calponin homology motif; EF, EF-hand motif; GAS2, GAS2 homology motif. (B) Sequence comparison of Kra with human Kra/BZAP45 (GenBank accession number BAA02795). Sequence identities (51%) are indicated by white letters on a black background, and sequence similarities (+1 or more in a PAM 250 matrix) are indicated by black letters on a gray background. N-terminal leucine zipper and C-terminal W2 domains are indicated above the sequences. Multiple alanine substitutions 12A and 7A, which were introduced into the AA-boxes 1 and 2, respectively, are also shown within the W2 domain. The boundaries of the kra cDNAs isolated from the yeast two-hybrid screen are marked by bent arrows. (C) Shot physically interacts with Kra. Soluble extracts of S2 cells transiently expressing HA-tagged Kra with Shot L(A)-GFP or C-Shot L-GFP were immunoprecipitated with IgG or anti-GFP antibody. The precipitates were subjected to SDS-PAGE and western blot analysis using anti-HA. (D) A region covering the EF-hand motifs of Shot is largely responsible for its interaction with Kra. In pull-down assays, GST-Kra was incubated with [35S]methionine-labeled C-terminal fragments of Shot containing AA residues 4688-5201 (C-Shot L), 4688-4915 (EF-GAS2), 4688-4858 (EF), 4859-4984 (GAS2) and 4916-5201 (EF-GAS2, an arrowhead). The relative input of the labeled proteins (20% of the amount used in each reaction) is shown in the bottom panel. (E) The AA-box 2 in Kra is essential for its interaction with Shot. Soluble extracts from S2 cells expressing C-Shot L-GFP with HA-KraWT, HA-Kra12A or HA-Kra7A were immunoprecipitated with IgG or anti-GFP. The presence of HA-Kra in the precipitates was detected by western blot analysis using anti-HA.

View larger version (90K): [in this window] [in a new window]   Fig. 2. Kra is expressed in the embryonic CNS. (A) Western blot analysis of S2 cell extracts using anti-Kra. The recognized protein is significantly depleted in kra RNAi-treated cells but not in gfp RNAi-treated control cells. (B-D) Whole-mount wild-type embryos stained with anti-Kra. Brackets mark the ventral nerve cord (VNC). (B) A stage 3 embryo. Lateral view. (C) A stage 13 embryo. Lateral view. (D) A stage 16 embryo. Ventral view. (E) A stage 16 embryo stained with preimmune serum. Ventral view. (F-H) A confocal section (1 µm) of a stage 16 wild-type embryo labeled with anti-Kra (F) and anti-Elav (G). (H) Merge of F and G. (I-K) A confocal section (1 µm) of a stage 16 wild-type embryo labeled with anti-Kra (I) and anti-Repo (J). (K) Merge of I and J. (I'-K') Higher magnification of the area marked with white broken lines in I-K. (L) The CNS of embryos, carrying both ap-GAL4 and UAS-HA-KraWT, stained with anti-HA. HA-Kra protein is detected in both the bodies (arrow) and the axons (arrowhead) of the Ap neurons. Scale bars: in F, 10 µm for F-L; in I', 10 µm for I'-K'.

View larger version (28K): [in this window] [in a new window]   Fig. 3. kra-null mutants. (A) Genomic organization of the kra locus. CG1427 and Vha26 genes are flanking the kra locus at position 83B4 on the right arm of the third chromosome. The relative positions of P-element insertions l(3)j9B6 and EP(3)0428 are indicated as inverted triangles. The extents of deletions in kra1 and kra2 are indicated. For the predicted transcripts, black boxes represent coding regions and white boxes represent untranslated regions; predicted translation initiation and stop sites are indicated. (B) Western blot analysis of larval extracts using anti-Kra. The same blot was reprocessed with anti-ß-actin to confirm equal protein loading. (C) RT-PCR analysis of CG1427 and rp49 expression in third instar wild-type and kra1/kra2 mutant larvae.

View larger version (66K): [in this window] [in a new window]   Fig. 4. kra and shot synergistically interact to affect CNS midline repulsion. (A-K) The CNS of embryos stained with mAb 1D4 (anti-Fasciclin II). (A-C) Early stage 13 embryos. (D-K) Stage 16-17 embryos. (A-I) Both kra and shot are required for midline axon repulsion. (A) In wild-type embryos, the pCC axon extends anteriorly and slightly away from the midline (arrow). (B,C) In kra1/kra2 (B) and shot3/shot3 (C) mutant embryos, the pCC axon abnormally crosses the midline (arrows). (D) The Fas II-positive axons show three parallel longitudinal pathways on each side of the midline in wild-type embryos. (E,G) The innermost pathway ectopically crosses the midline in kra1/kra2 (E) and shot3/shot3 (G) mutants. (F) The kra1/kra2 mutant phenotype is rescued by expressing the UAS-HA-KraWT transgene in all neurons. (H,I) The indicated transgenes are expressed in all neurons in shot3 as described (Lee and Kolodziej, 2002b). (H) The midline phenotype of shot is rescued by the UAS-Shot L(A)-GAS2-GFP transgene. (I) The UAS-Shot L(C)-GFP transgene fails to rescue the midline phenotype of shot. (J,K) Heterozygosity for kra and shot enhances the phenotypes of the shot and kra homozygotes, respectively, resulting in multiple crossing defects in shot3/+; kra1/kra2 (J) and shot3/shot3; kra2/+ (K) embryos. Anterior is to the top. (L) Quantification of midline crossing defects per segments in each of the indicated genotypes (wild-type, n=60 segments; shot3/+; kra2/+, n=240; kra1/kra2, n=504; C155-GAL4/+; kra1,UAS-HA-kraWT/kra2, n=440; shot3/shot3, n=272; shot3,1407-GAL4/shot3; UAS-Shot L(A)-GFP/+, n=192; shot3,1407-GAL4/shot3,UAS-Shot L(A)-GAS2-GFP, n=264; shot3,1407-GAL4/shot3,UAS-Shot L(A)- EF-GFP, n=640; shot3,1407-GAL4/shot3,UAS-Shot L(C)-GFP, n=816; shot3/+; kra1/kra2, n=184; shot3/shot3; kra2/+, n=120; shot3/shot3; kra1/kra2, n=298). Statistically significant differences, as determined by Student's t-test, are denoted by an asterisk (control, wild-type; P<0.001), double asterisk (control, kra1/kra2; P<0.017) or triple asterisk (control, shot3/shot3; P<0.001). Scale bar: 10 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 5. kra and shot genetically interact with robo. (A-F) The CNS in stage 16-17 embryos stained with mAb 1D4 (anti-Fasciclin II). In kra2/+ (A) or shot3/+ (B) heterozygous embryos, the longitudinal axon tracts never cross the midline as in the wild-type (see Fig. 4D). (C) In robo2/+ heterozygous embryos, the longitudinal axon tracts rarely cross the midline (0.9%). (D,E) The robo2/+ phenotype is significantly enhanced when one copy of kra and shot is also removed in robo2/+; kra2/+ (D) or shot3,robo2/+ (E) transheterozygous embryos. (F) This phenotype is also further enhanced in shot3,robo2/+; kra2/+ embryos. Anterior is to the top. (G) Quantification of the midline crossing frequency per segments in each of the indicated genotypes (n=180, 130, 446, 392, 976 and 488 segments, respectively). Statistically significant differences are denoted by an asterisk (control, robo2/+; P<0.001). Scale bar: 10 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 6. Robo-positive axons cross the CNS midline in kra mutant embryos. (A-F) Stage 16 embryos carrying the apC-tau-lacZ marker were doubly stained with anti-Slit (A,D) and anti-ß-galactosidase (B,E). (A-C) A wild-type embryo. (D-F) A kra1/kra2 mutant embryo; some Ap axons ectopically cross the midline (arrowheads in E,F). (G-L) Stage 16 embryos carrying the apC-tau-lacZ marker were doubly stained with anti-Robo (G,J) and anti-ß-galactosidase (H,K). (G-I) A wild-type embryo. (J-L) A kra1/kra2 mutant embryo; Robo is abnormally detected in the commissural tracts that contain Ap axons crossing the midline (arrowheads in K,L). Scale bar: 10 µm.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Kra is a translation inhibitor. (A) Kra cosediments with the 40S ribosomal subunit in a sucrose gradient of S2 cell extracts. Cell extracts were prepared in the presence of 100 µg/ml cycloheximide and fractionated on a 7-47% sucrose gradient. Fractions were analyzed for absorbance at 260 nm and subjected to SDS-PAGE and western blot analysis using anti-Kra, anti-eIF4E and anti-L28 antibodies. Positions of 40S and 60S ribosomal subunits, 80S ribosomes, and polysomes are indicated. (B) Kra binds eIF2ß in vitro. An SDS-gel stained with Coomassie Blue shows GST, GST-eIF5 and GST-Kra proteins in the amounts used for pull-down assays (top panel). A recombinant GST-Kra fusion protein, but not GST alone, interacts with [35S]methionine-labeled eIF2ß (bottom panel). GST-eIF5 was used as a positive control for binding. (C) The AA-boxes in the W2 domain of Kra are important for its efficient binding to eIF2ß in vivo. HA-tagged Kra-WT, -12A, or -7A was coexpressed with GFP-tagged eIF2ß in the Drosophila embryo. Embryo extracts were immunoprecipitated with anti-HA, and the precipitated eIF2ß was detected by western blot analysis using anti-GFP. (D) Shot and eIF2ß can simultaneously bind to Kra. Soluble extracts of S2 cells overexpressing C-Shot L-GFP were immunoprecipitated with anti-GFP or IgG, and the presence of endogenous Kra and eIF2ß in the precipitates was determined by western blot analysis using either anti-Kra or anti-eIF2ß antibody, respectively. (E) Kra inhibits translation in vitro. In the top panel, luciferase mRNA was translated in reticulocyte lysates with [35S]methionine in the absence and presence of the indicated proteins (2.4 µM); reaction samples were analyzed by SDS-PAGE and autoradiography. Translation of luciferase mRNA is specifically inhibited by the addition of Kra. In the middle panel, 10% of each reaction was removed after incubation and analyzed by RT-PCR for the presence of luciferase mRNA. In the bottom panel, the indicated amounts of GST, Kra-WT and Kra-7A were added to reticulocyte lysates. Cyc, cycloheximide (100 µg/ml).

View larger version (18K): [in this window] [in a new window]   Fig. 8. The Kra AA-boxes 1 and 2 are essential for Kra functionality in vivo. When expressed under the control of the panneuronal driver C155-GAL4, neither UAS-HA-kra12A nor UAS-HA-kra7A rescues the kra1/kra2 mutant phenotype at the midline (n=312 and 360, respectively). Note that UAS-HA-kraWT significantly attenuates the midline crossing phenotype (see Fig. 4F,L).

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