Figure 4. Staufen interferes with... | Rockefeller University Press

Figure 4.

$Staufen interferes with association of Egl with oskar RNPs in the oocyte. (A and A′) Staufen-GFP (green) distribution in the nurse cell cytoplasm (left-hand images) and oocyte (right-hand images) before (stage 6–7, A) and during (stage 8–9, A′) the posterior localization of endogenous oskar mRNA (magenta) in the oocyte of the same egg chamber. (B and B′) Quantification of Staufen-GFP association with oskar RNPs in nurse cell cytoplasm and ooplasm at the indicated stages as a function of RNA copy number. The number of analyzed oskar RNPs was 3,105 (stage 5–7 nurse cells), 8,749 (stage 9 nurse cells), 3,814 (stage 5–7 oocytes) and 14,054 (stage 9 oocytes). (C and C′) Distribution of Egl-GFP (green) in the nurse cell cytoplasm (left-hand images) and oocyte (right-hand images) before (stage 7, C) and during (stage 9, C′) the posterior localization of endogenous oskar mRNA (magenta) in the oocyte of the same egg chamber. In A, A′, C, and C′, nurse cell images are shown with different brightness/contrast settings for better visualization of the signals. Cyan arrows point to examples of colocalization. Scale bars represent 10 μm. (D and D′) Quantification of association of Egl-GFP with oskar RNPs in nurse cell cytoplasm and ooplasm at the indicated stages. The number of analyzed oskar RNPs was 8,511 (stage 5–7 nurse cells), 10,210 (stage 9 nurse cells), 6,897 (stage 5–7 oocytes), and 15,869 (stage 9 oocytes). (E–F′) Association of Egl-GFP with oskar RNPs in the stage 9 oocyte in the indicated genotypes. In A–B′, endogenous Staufen was absent (stauR9/stauD3 background), whereas in C–F′ endogenous, unlabeled Egl was present. 15,869–26,232 oskar RNPs were analyzed for each genotype. Error bars represent 95% confidence intervals in B, B′, and D–F′. In D and E, datapoints are slightly offset in the x-axis to facilitate comparison. The size of the circles is proportional to the relative abundance of each category of oskar RNPs within the overall population. Triangles indicate that the fraction of GFP-positive oskar RNPs is not significantly different from zero (P > 0.01, one sample t test, B, B′, and D–F′). In B′, D′, E′, and F′, GFP protein intensities on oskar RNPs in the oocytes are normalized to GFP signal intensities in the corresponding nurse cells. (G) Fold-enrichment of oskar mRNA precipitated from UV crosslinked Egl-GFP ovarian extracts relative to the GFP control in control and stau RNAi. Different colors represent different experiments (four in total). Solid (P < 0.05) or dashed lines (P > 0.05) connect paired data (pairwise Student’s t test was used to determine significance). Empty triangles indicate non-significant enrichment (P > 0.05) of oskar relative to the GFP control. Error bars show SD of three replicates per experiment.$

Staufen interferes with association of Egl with oskar RNPs in the oocyte. (A and A′) Staufen-GFP (green) distribution in the nurse cell cytoplasm (left-hand images) and oocyte (right-hand images) before (stage 6–7, A) and during (stage 8–9, A′) the posterior localization of endogenous oskar mRNA (magenta) in the oocyte of the same egg chamber. (B and B′) Quantification of Staufen-GFP association with oskar RNPs in nurse cell cytoplasm and ooplasm at the indicated stages as a function of RNA copy number. The number of analyzed oskar RNPs was 3,105 (stage 5–7 nurse cells), 8,749 (stage 9 nurse cells), 3,814 (stage 5–7 oocytes) and 14,054 (stage 9 oocytes). (C and C′) Distribution of Egl-GFP (green) in the nurse cell cytoplasm (left-hand images) and oocyte (right-hand images) before (stage 7, C) and during (stage 9, C′) the posterior localization of endogenous oskar mRNA (magenta) in the oocyte of the same egg chamber. In A, A′, C, and C′, nurse cell images are shown with different brightness/contrast settings for better visualization of the signals. Cyan arrows point to examples of colocalization. Scale bars represent 10 μm. (D and D′) Quantification of association of Egl-GFP with oskar RNPs in nurse cell cytoplasm and ooplasm at the indicated stages. The number of analyzed oskar RNPs was 8,511 (stage 5–7 nurse cells), 10,210 (stage 9 nurse cells), 6,897 (stage 5–7 oocytes), and 15,869 (stage 9 oocytes). (E–F′) Association of Egl-GFP with oskar RNPs in the stage 9 oocyte in the indicated genotypes. In A–B′, endogenous Staufen was absent (stau^R9/stau^D3 background), whereas in C–F′ endogenous, unlabeled Egl was present. 15,869–26,232 oskar RNPs were analyzed for each genotype. Error bars represent 95% confidence intervals in B, B′, and D–F′. In D and E, datapoints are slightly offset in the x-axis to facilitate comparison. The size of the circles is proportional to the relative abundance of each category of oskar RNPs within the overall population. Triangles indicate that the fraction of GFP-positive oskar RNPs is not significantly different from zero (P > 0.01, one sample t test, B, B′, and D–F′). In B′, D′, E′, and F′, GFP protein intensities on oskar RNPs in the oocytes are normalized to GFP signal intensities in the corresponding nurse cells. (G) Fold-enrichment of oskar mRNA precipitated from UV crosslinked Egl-GFP ovarian extracts relative to the GFP control in control and stau RNAi. Different colors represent different experiments (four in total). Solid (P < 0.05) or dashed lines (P > 0.05) connect paired data (pairwise Student’s t test was used to determine significance). Empty triangles indicate non-significant enrichment (P > 0.05) of oskar relative to the GFP control. Error bars show SD of three replicates per experiment.