DEV027698 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Oskar interacts specifically with the SH3 domain of Lasp. (A) Short-Oskar fused to the N terminus of NLS-LexA, but not LexA fused to the M1M2 region of Oskar, interacts with clone 3.30 in a two-hybrid assay. Clone 3.30 represents one of five independent activator-cDNA fusion classes encoding the C-terminal portions of Lasp that were identified in our two-hybrid screen. The SH3 domain encoded by the C terminus of clone 3.30 is required (WrA) and sufficient (Suf) for the interaction to occur. The interaction is highly specific, as Short-Oskar fused to LexA does not interact with the SH3 domains of any of the following: the mouse EGF receptor substrate EPS8; chicken Src; human Ras GAP; the p85 subunit of bovine phosphatidylinositol 3-kinase; or human phospholipase Cγ. (B) Western blot analysis demonstrates that clone 3.30, the point mutant WrA, and the SH3 domain of clone 3.30 alone (Suf) are expressed at similar levels in yeast. The fusion proteins were detected using a monoclonal antibody that recognizes the HA epitope preceding the cDNA inserts. (B) The interaction of full-length Long- and Short-Oskar with clone 3.30 depends on bait expression (Oskar is under control of the gal promoter which is repressed on glucose). (D) Pull-downs with Gst-fusion proteins, showing that only full-length Lasp and clone 3.30 with an intact SH3 domain precipitate Long- and Short-Oskar from ovary extracts above background (left panel). The right panel shows the Coomassie-stained gel corresponding to the autoradiograph shown on the left. (E) Identity scores of Drosophila Lasp with human, Mouse, Rat, Rabbit and C. elegans Lasp are given in percent for each of the domains. Neither the length nor the sequence of the spacer is conserved.

• Supplemental Figure S2 -

  Fig. S2. Cortical localization of Lasp depends on actin. (A) In untreated ovaries, actin (A′) and Lasp (A′′) colocalize at the cell cortex. (B) Treatment with Latrunculin A (200 µM for 20 minutes) leads to the destabilization of F-actin (B′) and the concomitant loss of cortical Lasp localization (B′′). Stage 9 oocytes are shown.

• Supplemental Figure S3 -

  Fig. S3. oskar mRNA levels are not changed in embryos laid by lasp mutant mothers. RT-PCR quantification of oskar mRNA normalized to rp49 mRNA shows that the loss of oskar RNA from the posterior pole of lasp^y41 or lasp^y45 embryos is not due to an overall loss of oskar mRNA, but specifically to a redistribution from the posterior pole.

• Supplemental Figure S4 -

  Fig. S4. The total amount of Oskar protein is reduced in osk⁵⁴ heterozygotes when no Lasp is present. (A,B) Western blot analysis of increasing amounts of ovary lysate from the indicated genotypes probed for (A) Oskar (L-Osk and S-Osk) and Tubulin, or (B) Oskar and Kinesin heavy chain (KHC) as a normalization control for the genotypes indicated above. (C) Quantification on a calibrated GS-800 densitometer showed that removal of lasp reduces the amount of Short-Oskar by 40% (normalized to Tubulin) or 50% (normalized to KHC) in osk⁵⁴ heterozygotes.