Localization-Dependent Oskar Protein Accumulation Arie Koen Braat, Nan Yan, Eric Arn, Dianne Harrison, Paul M Macdonald  Developmental Cell  Volume 7, Issue 1, Pages 125-131 (July 2004) DOI: 10.1016/j.devcel.2004.06.009

Figure 1 Loss of NAC Activity Disrupts Localization of osk mRNA (A and B) Tight posterior localization of Stau protein (which colocalizes with osk mRNA) to the posterior pole of wild-type stage 9 and 10 oocytes. (C and D) Distribution of Stau in large bodies in aic germline clones at different stages. (E and F) Similar large bodies in bic germline clones, with a high magnification view (F) showing that the bodies appear roughly circular in this focal plane, and are primarily spherical in shape. (G) Revertant of the aic mutant, with a normal pattern of Stau protein. (H and I) The large bodies, identified by Stau staining in (H), do not contain ER proteins, as shown by the absence of specific KDEL staining in (I). Developmental Cell 2004 7, 125-131DOI: (10.1016/j.devcel.2004.06.009)

Figure 2 Loss of NAC Activity Affects Osk Protein Accumulation (A) Distribution of Osk protein in a wild-type oocyte. In mutant germline clones (aic, [B and C]; bic, [D and E]), Osk is either roughly wild-type in distribution (D) or is more dispersed along the cortex (B, C, E). Examination of more than 30 stage 9 or 10 oocytes for each mutant reveals no obvious differences in the nature of their defects. Developmental Cell 2004 7, 125-131DOI: (10.1016/j.devcel.2004.06.009)

Figure 3 Distribution of mRNAs in Polysome Gradients (A) RNAs in the cytoplasmic fraction detected by RNase protection assays. The three mRNAs, osk, α tubulin, and rp49, were detected in the fractions from the same set of two gradients, one to detect polysomes and one to which puromycin was added to disrupt polysomes. Both osk and α tubulin mRNAs are present at high levels in the polysome peak fractions (close to the bottom of the gradient) and are largely lost from those fractions when polysomes are disrupted by addition of puromycin. Using the method of calculation described for Figure 4D, 35% of osk mRNA is polysomal. The rp49 mRNA is not significantly associated with polysomes (and thus was not examined following addition of puromycin). Two of the samples used for detection of rp49 mRNA were lost during the assay (second and final lanes), but their absence does not affect the interpretation of the experiment. (B) Distribution of osk mRNA in the detergent-solubilized fraction. As in the soluble fraction, osk is readily detected in polysomes, and this association is dramatically reduced when puromycin is used to disrupt the polysomes. Using the method of calculation described for Figure 4D, 57% of osk mRNA is polysomal. Similar assays for α tubulin mRNA were performed, but the levels of that mRNA in the detergent-solubilized fraction were too low to detect by RNase protection. Developmental Cell 2004 7, 125-131DOI: (10.1016/j.devcel.2004.06.009)

Figure 4 Polysomal Association of osk mRNA in the Absence of Osk Protein Accumulation Detection of osk mRNA in the cytoplasmic (A) and detergent-solubilized (B) fractions of btz1/Df(3R)IR16 (labeled btz−) and stauD3 P[StauΔdsRBD5]/stauD3 (labeled stau−) ovaries. For both mutants, osk mRNA remains largely in the polysomal part of the gradient, and the association with polysomes is confirmed by the shift upwards in the gradient (toward the left of the figure) in the presence of puromycin. (C) Western blot analysis of Osk. Lane 1, osk54/Df(3R)pXT103; lane 2, wt; lane 3, stauD3 P[StauΔdsRBD5]/stauD3. Osk protein is detected in the upper panel, with the two isoforms, Long Osk and Short Osk, indicated. Tubulin was detected in the lower panel and serves as a loading control. Approximately 5% of the wt level of Osk persists in the stauD3 P[StauΔdsRBD5]/stauD3 mutant, as estimated by diluting the wt extract and comparing to the mutant extract. (D) Quantitation of osk mRNA in polysomal fractions in the stauD3 P[StauΔdsRBD5]/stauD3 mutant. The levels of osk mRNA in individual fractions of the polysome gradients from (A) and (B) were quantitated by phosphorimager analysis and presented as percentages of total osk mRNA in the gradient. Open circles indicate osk mRNA when polysomes are intact, and closed circles indicate osk mRNA when polysomes are disrupted by addition of puromycin. To estimate what fraction of osk mRNA in each panel is polysomal, the fraction of osk mRNA shifted out of the polysomal region by the addition of puromycin (dark gray areas) was divided by the total osk mRNA of the sample not treated with puromycin (light and dark gray areas). For the soluble fraction, 54% of osk mRNA was estimated to be polysomal, and for the detergent-solubilized fraction, 38% of osk mRNA was estimated to be polysomal. Developmental Cell 2004 7, 125-131DOI: (10.1016/j.devcel.2004.06.009)