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Figure 2. Heterotrimeric Kinesin II Is Required for Localization of Vg1 mRNA(A–C) Stage III/IV oocytes were preinjected with PBS (A), mouse IgG (B), or the K2.4 monoclonal heterotrimeric function-blocking antibody (C) and assayed for Vg1 mRNA localization by in situ hybridization to the injected XβG-VgLE fusion transcript. (D–F) Stage III/IV oocytes were injected with the XβG-VgLE RNA alone (D) or coinjected with the XβG-VgLE and AUG− mutant (E), or the Xklp3b dominant-negative mutant mRNA (F). (G) Western-blot analysis (using an anti-HA specific antibody) of oocytes injected with the HA-tagged Xklp3b dominant-negative construct (right) or the AUG mutant (left). A protein fragment of approximately 50 kDa was detected in extracts prepared 3–6 hr after injection of the Xklp3b dominant-negative RNA but not in extracts prepared from oocytes injected with the AUG− mutant RNA. (H and I) Oocytes coinjected with digoxygenin-labeled Xcat-2 LE and the AUG− mutant (H) or the Xklp3b dominant-negative mRNA (I). Localization frequencies of the Xcat-2 LE were 89% (n = 37) for the AUG− mutant and 23% (n = 35) for the dominant-negative construct. The scale bar represents 100 μm in (A)–(F) and 500 μm in (H) and (I). |
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Figure 4. Colocalization of Kinesin II and RNA in the Ring Structure at the Vegetal Pole(A) In situ hybridization to endogenous Vg1 mRNA in a stage II oocyte showing the RNA localizing to a ring structure (arrow) at the vegetal pole.(B) Injected digoxygenin-labeled Xcat-2 LE localizes to a similar structure.(C) Optical cross-section of the wedge and ring structure from a stage II oocyte injected with alexa fluor 546-labeled Xcat-2 LE (red) and detection of endogenous kinesin II as in Figure 3. The most extensive overlap in labeling occurs at the extremity of the wedge (yellow) in the cortex, corresponding to a ring structure (arrows) at the vegetal pole. Note that kinesin II recruited to the wedge appears to travel deeper into the cortex than the RNA. The vegetal pole is oriented toward the bottom right of each panel in (C). The scale bar represents 100 μm in (A) and (B) and 10 μm in (C). |
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gdf1 (growth differentiation factor 1) gene expressionin Xenopus laevis, assayed by in situ hybridization in oocyte stage II, vegetal pole down. (GV = germinal vesicle). |
