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Fig. 2. Celf1 is a cytoplasmic protein that co-localizes with vegetal RNAs and known localization factors in Xenopus oocytes. (A) Temporal analysis of Celf1 expression during Xenopus oogenesis and embryogenesis. Western blot analysis of Celf1 with equivalent amounts of oocyte and embryonic extract; stages of oogenesis and embryogenesis were as indicated. (B) Western blot analysis of Celf1 with cytoplasmic (C) and nuclear (N) fractions from staged oocytes. α-Stau1 was utilized as control for a cytoplasmic ( Allison et al., 2004) and α-Hnrnpab for a predominantly nuclear oocyte protein ( Czaplinski et al., 2005). (C) Co-localization analysis for endogenous Celf1 protein and vegetally localizing, microinjected Cy3-dnd1-LE RNA in Xenopus oocytes. α-Celf1 immunostaining was performed on Cy3-dnd1-LE RNA injected stage III oocytes. (D) α-Celf1/α-Igf2bp3 co-immunostaining was performed with stage III oocytes that were injected with Cy3-dnd1-LE. Single confocal sections are shown. Scale bars indicate 100 µm (whole oocyte) and 20 µm (magnification). |
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Fig. 5. Efficient binding of Celf1 to dnd1-LE RNA is critical for vegetal RNA localization. Wild-type and the mutant dnd1-LE with reduced affinity for Celf1 were analyzed for their ability to localize to the vegetal cortex in Xenopus oocytes. (A) Nuclei of stage III oocytes were injected with Cy3-labeled wild-type (wt) or mutant (mut) dnd1-LE RNA, fixed after 0–48 h, and analyzed by confocal imaging. Single confocal sections are shown. The scale bar indicates 100 µm. (B) Regional RNA quantification was done by measuring the ratio of the mean fluorescence along a line at the vegetal cortex (Vg) versus a line in the animal cytoplasm (An). Kinetics of localization in a time span of 5 to 48 h after microinjection of mutant (mut) or wild-type (wt) dnd1-LE RNA. Error bars indicate standard deviation of approx. 10 oocytes. (C) Quantification of localization efficiency for wild-type (wt) and mutant (mut) dnd1-LE. Stage III oocytes were co-injected with wild-type (wt) or mutant (mut) Cy3- or Atto633-labeled dnd1-LE RNAs. Average localization efficiencies of three independent experiments are shown. The error bars indicate standard error of the mean. p<0.05 is indicated (*). |
