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First published online 13 September 2006
doi: 10.1242/dev.02570

Development 133, 3963-3972 (2006)
Published by The Company of Biologists 2006
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The {gamma}TuRC components Grip75 and Grip128 have an essential microtubule-anchoring function in the Drosophila germline

Nina Vogt1,*, Iris Koch1, Heinz Schwarz2, Frank Schnorrer1,{dagger} and Christiane Nüsslein-Volhard1

1 Max-Planck-Institute for Developmental Biology, Department of Genetics, Spemannstr. 35, 72076 Tübingen, Germany.
2 Max-Planck-Institute for Developmental Biology, Electron Microscopy Unit, Spemannstr. 35, 72076 Tübingen, Germany.


Figure 1
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  		Fig. 1. Molecular characterization of Grip128 mutant alleles. (A) Schematic overview of the Grip128 protein and the mutant alleles. Motifs are indicated (Gunawardane et al., 2000Go). C to T changes result in STOP codons replacing Gln662 in Grip128326 and Gln706 in Grip128352. (B) Wild-type and homozygous mutant ovarian and male extracts were analyzed by western blotting with a Grip128 antibody (Gunawardane et al., 2000Go). Grip128 is indicated by an arrow. Additional bands are non-specific.

 

Figure 2
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  		Fig. 2. bcd RNA localization requires Grip128. (A-F) Wild-type (A-C) and Grip128326 mutant (D-F) oocytes at stages 9 (A,D) and 11 (B,E), and 0- to 30-minute-old eggs (C,F) were stained for bcd RNA. (G,H) Wild-type (G) and Grip128326 mutant (H) oocytes at stage 11 were stained for microtubules. The anterior microtubule subset is indicated by an arrowhead and the subcortical microtubules by arrows. (J,K) Stage-11 wild-type (J) and Grip128326 mutant (K) oocytes carrying the NZ transgene were stained for the ß-galactosidase reporter.

 

Figure 3
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  		Fig. 3. Analysis of the ovarian {gamma}TuRC. (A) Expression analysis of {gamma}TuRC components and Swa in ovarian extracts of the indicated genotypes. (B) Anti-{gamma}Tub37C-immunoprecipitation from ovarian extracts of the indicated genotypes. (C-E) Wild-type (C), Grip75175 (D) and Grip128326 (E) homozygous mutant ovarian extracts were fractionated by 5-40% sucrose density gradient centrifugation. The first lane in each panel corresponds to the input, whereas subsequent lanes contain fractions obtained from top to bottom of the gradient.

 

Figure 4
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  		Fig. 4. Meiotic defects in Grip75 and Grip128 mutant eggs. (A-C) DAPI staining of wild-type (A), Grip75175 (B) and Grip128326 mutant (C) stage 14 oocytes. The chromosome arrangement is shown during metaphase arrest. (D-F) Meiosis I spindles in wild-type (D), Grip75175 (E) and Grip128326 mutant (F) eggs. {alpha}-Tubulin is shown in green and DNA in red. (G-J) Meiosis II spindles in wild-type (E), Grip75175 (H) and Grip128326 mutant (J) eggs, stained as in D-F. The arrow indicates the central MTOC in wild type. Confocal stacks were merged into single images.

 

Figure 5
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  		Fig. 5. Meiosis is perturbed in Grip75 and Grip128 mutant spermatocytes. (A-C) Wild-type (A), Grip75175 (B) and Grip128352 (C) mutant spermatids at the onion stage. Nuclei appear bright in phase-contrast microscopy (arrowheads), whereas mitochondria are dark (arrows). (D-F) EM cross-sections of wild-type (D), Grip75175 (E) and Grip128352 (F) mutant sperm flagella. Scale bars: 500 nm. The arrowheads indicate axonemes, the arrows indicate mitochondria. (G-J) Meiosis I spindles at metaphase in wild-type (G), Grip75175 (H) and Grip128352 (J) mutant spermatocytes. Microtubules are shown in green, DNA is in red.

 

Figure 6
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  		Fig. 6. Grip75 and Grip128 anchor microtubules at the nucleus. (A-C) Wild-type (A), Grip75175 (B) and Grip128352 (C) mutant sperm were stained for {alpha}-tubulin (green) and DNA (red). Nuclei are located at one end of a sperm bundle in wild-type testes but are dispersed along the bundle in mutant testes. (D-F) Wild-type (D), Grip75175 (E) and Grip128352 (F) mutant elongating spermatids were stained for {gamma}-tubulin (green) and DNA (red). In wild-type spermatids, {gamma}-tubulin is associated with the nucleus, whereas this association is frequently lost in mutant spermatids (arrows). (G-M) EM cross-sections of wild-type (G,K), Grip75175 (H,L) and Grip128352 (J,M) mutant axonemes in early (G-J) or late (K-M) stages of axoneme assembly. Scale bars: 100 nm.

 




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