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First published online 26 November 2008
doi: 10.1242/dev.027698

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The actin-binding protein Lasp promotes Oskar accumulation at the posterior pole of the Drosophila embryo

Ritsuko Suyama^*, Andreas Jenny^*,, Silvia Curado^*,, Wendy Pellis-van Berkel and Anne Ephrussi^¶

Developmental Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.

View larger version (42K): [in this window] [in a new window]   Fig. 1. Lasp interacts with Oskar in vitro, and binds and colocalizes with actin. (A) Gst pull-down assays show that [35S]-labeled Short-Oskar binds to a Gst fusion-protein of the original two-hybrid clone (lane 6, Gst_3.30) and of the SH3 domain of Lasp (lane 8, Gst_Suf). A functional SH3 domain is required for this interaction, as the WA point mutant version that abolishes physiological SH3 interactions is unable to bind (lane 7, Gst_WA). Neither Gst fused to the SH3 domain of Src (lane 4), nor Gst alone (lane 5) retains Oskar. However, the Src substrate SAM68 binds to the Src SH3 domain but not to its natural, neuronal, non-binding variant NSrc (lanes 12 and 11, respectively), showing that the SH3 domain of Src is functional. Lanes 2 and 10 contain 10% of the respective labeled proteins added to the binding reactions. The apparent molecular masses of the standards (lanes 1 and 9) are indicated in kDa. (B) Coomassie-stained gel showing that comparable amounts of the indicated fusion protein were bound to the glutathione-Sepharose beads. (C) Lasp binds to filamentous actin in vitro. The N terminus of Lasp, including the two NEB repeats fused to Gst (GN) co-sediments with F-actin (Ac) in pelletting assays (P, pellets) of duplicate reactions in lanes 5 and 6, supernatants (S) in lanes 3 and 4). Lanes 1 and 2 show that hardly any fusion protein is pelletted in the absence of actin. The position of the 45-kDa molecular mass marker is indicated on the right. (D-F) nanos-Gal4 driven, germline-specific expression of GFP-tagged full-length Lasp in a stage 8 egg chamber. Lasp (D) and actin (rhodamine phalloidin; E) colocalize around the oocyte cortex, at the nurse cell borders and on ring canals (F). DNA is stained in blue (DAPI).

View larger version (57K): [in this window] [in a new window]   Fig. 2. Lasp and Oskar overlap at the posterior pole of the oocyte. (A) In the germarium, antibodies against Lasp strongly and specifically stain wild-type somatic cap cells and cysts in region 2, as the staining is absent in laspy41 mutants (A'; inset in A' shows DAPI staining of the mutant germarium). (B) Stage 6 egg chamber. Lasp is strongly expressed in the nurse cells and is enriched at the posterior of the oocyte. The anterior (not in the focal plane in this image) and posterior polar follicle cells are stained as well. Lasp is mainly found around cell cortices. A stained ring canal is indicated with an arrowhead. (C) At stage 10, Lasp stains the periphery of nurse cells, ring canals (arrowheads) and the oocyte cortex. Weaker staining can also be detected in the follicle cells. (D-F) In a stage 10 oocyte, Lasp (D) and Oskar (E) colocalize at the posterior pole (F: Lasp, green; Oskar, red; DNA, blue). The inset in F shows an intensity profile across the oocyte posterior pole (region of the white square). The fluorescence intensities of both profiles peak at a similar position at the posterior cortex and demonstrate the overlapping localization.

View larger version (93K): [in this window] [in a new window]   Fig. 3. Lasp in early embryos colocalizes with Oskar and F-actin. Confocal images of Lasp, Oskar and F-actin distribution in early embryos. (A-D) Lasp and Oskar overlap at the posterior pole of a wild-type preblastoderm stage embryo (A). (B-D) Magnification of the posterior tip of the embryo shown in A, stained with anti-Lasp (B) and anti-Oskar (C); merged image in D. (A'-D') Corresponding stainings of a laspy41 mutant embryo. No Lasp signal is detected in the mutant, demonstrating that antibody is specific. (E-J) Although simultaneous detection of Lasp and F-actin was not possible for technical reasons, Lasp distribution parallels that of F-actin, extending along the network of actin fibers throughout the embryo. At the syncytial blastoderm stage, both Lasp and actin are localized apically (E-G). At the blastoderm stage, Lasp is localized apically and laterally within the epithelial cell; F-actin is distributed apically, laterally and basally (H-J). (H',I') Lasp staining of a laspy41 mutant blastoderm embryo, again demonstrating the specificity of the antibody.

View larger version (44K): [in this window] [in a new window]   Fig. 4. Generation and characterization of lasp loss-of-function mutants. (A) Domain organization of Drosophila Lasp. S-Lasp (55 kDa) lacks sequences corresponding to the beginning of the fifth exon to 474-bp downstream of fifth exon of L-Lasp (80 kDa). (B) Diagram of the genomic region of lasp. Color code of lasp exons is as in A. CG9692, a predicted intronic gene (pink), is located between the first and the second exons of lasp. P{wHy}DG14505 inserted in the second intron of lasp was remobilized to obtain deletions in the locus (laspy45 and laspy41; indicated by black bars). (C) RT-PCR on wild-type (wt) ovarian and embryonic RNA. The RT-PCR products specific for L-Lasp (upper band, 700 bp) and S-Lasp (lower band, 220 bp) were generated using the primers indicated in B. Note that a low level of the short isoform is detected in the ovary only. -, no reverse-transcriptase control. (D) Western analysis demonstrates the absence of Lasp in the deletion mutants laspy41 and laspy45. In the wild-type (wt) ovary extract (left panel), the antibody recognizes a major and minor band of 80 and 55 kDa corresponding to full-length L-Lasp and S-Lasp, as identified by comparison with maternal-tubulin driven L- and S-Lasp in a lasp- background (right panels). In addition, a possible degradation product of L-Lasp of 75 kDa is seen. Consistent with the RT-PCR data, in the wt embryo extract (middle panel), the antibody recognizes a single band of 80 kDa corresponding to L-Lasp. No Lasp protein is seen in the deletion mutants laspy41 and laspy45 [using antisera raised against the C terminus or N terminus (not shown)] of Lasp, confirming that they are null mutants. Tubulin was used as loading control (lower panels).

View larger version (44K): [in this window] [in a new window]   Fig. 5. Oskar levels are reduced in lasp mutant embryos. (A) Stage 10 wild-type (wt) and lasp mutant egg chambers. Lasp (red) is localized around the oocyte cortex and Oskar (green) is localized at the posterior pole (upper panel). Lasp is not detected in the lasp mutant, but Oskar is localized at the posterior as in wild type (lower panel). (B) Western blot detection of Oskar protein in extracts of wild-type and lasp mutant ovaries and embryos (as indicated on top of the panels). Levels of Short-Oskar as well as the N-terminal extension containing Long-Oskar (arrows) are reduced in the lasp mutant compared with in wild-type ovaries and embryos. -Tubulin was used as loading control (lower panels). (C) Compared with bcd mRNA at the anterior (bottom panels), Oskar protein (top) and mRNA (middle panels) levels are reduced at the posterior of lasp mutant embryos. For comparison, wild-type embryos are shown on the left. (D) Quantification of effect shown in C. Comparison of fluorescent signal intensities showed that the reduction of Oskar protein and mRNA is specific and significant at P<0.001 (***).

View larger version (72K): [in this window] [in a new window]   Fig. 6. lasp interacts genetically with oskar. (A) The grandchildless phenotype (absence of ovaries) was quantified by dissecting female offspring of the indicated genotypes. laspy45, osk54/laspy45 females display a strong grandchildless phenotype compared with laspy45, osk54/+ females (***statistically significant at P<0.001; 2 test). (B,C) Pole cells of stage 11 embryos produced by females of the indicated genotypes were stained with anti-Vasa antibodies (C) and counted (B). The number of pole cells in progeny of laspy45, osk54/laspy45 (12 cells on average) was half that of the osk54/+ progeny (23 cells on average; ***P<0.001; two-sample t-test). Wild-type embryos have an average of 33 pole cells.

View larger version (64K): [in this window] [in a new window]   Fig. 7. The Lasp SH3 domain is required for Lasp-Oskar interaction in vivo. (A) pCOG driven L-Lasp rescues the grandchildless phenotype of laspy45, osk54/laspy41 females (+/Cyo; laspy45, osk54/laspy41, baseline; **P<0.01; 2 test). Neither the non-Oskar-binding SH3 variant of L-Lasp (L-LaspWA) nor S-Lasp was able to rescue the grandchildless phenotype, suggesting an essential role for the Lasp-Oskar interaction in vivo. (B,C) Examples of stage 11 embryos laid by mothers of the indicated genotypes stained with anti-Vasa antibodies to visualize the pole cells (B). The reduced pole cell number of lasp y45, osk54/laspy41 embryos was partially rescued by pCOG L-Lasp (C; 25 cells on average; *P<0.05; two-sample t-test), but not by pCOG L-Lasp (WA) nor pCOG S-Lasp, again suggesting an in vivo relevance of a direct Oskar-Lasp interaction. Similar results were obtained using UAS-Lasp transgenes driven by maternal tubulin-Gal4VP16 or pCOG Gal4Vp16 (data not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 8. Lasp regulates tight localization of Oskar in the embryo. (A) Hatch rate of embryos and cuticle analysis of unhatched embryos with defects produced by wild-type (wt) or lasp mutant females expressing the osk-K10 3'UTR transgene under maternal tubulin-Gal4 control. Removal of lasp reduces the phenotype of ectopic Oskar. (B) Representative cuticle examples of - top to bottom - wild-type, head-defect, mild and strong bicaudal and shapeless classes of defects scored in A. (C) Pole cell staining of cellular blastoderm-stage embryos laid by wild-type or lasp mutant females, respectively, expressing the osk-bcd 3'UTR as indicated. Pole cells are stained with anti-Vasa antibody. The removal of lasp reduces the percentage of embryos with ectopic, anterior pole cells (as indicated above each panel).

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