XB-ART-4014Curr Biol February 3, 2004; 14 (3): 219-24.
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Kinesin II mediates Vg1 mRNA transport in Xenopus oocytes.
The subcellular localization of specific mRNAs is a widespread mechanism for regulating gene expression. In Xenopus oocytes microtubules are required for localization of Vg1 mRNA to the vegetal cortex during the late RNA localization pathway. The factors that mediate microtubule-based RNA transport during the late pathway have been elusive. Here we show that heterotrimeric kinesin II becomes enriched at the vegetal cortex of stage III/IV Xenopus oocytes concomitant with the localization of endogenous Vg1 mRNA. In addition, expression of a dominant negative mutant peptide fragment or injection of a function-blocking antibody, both of which impair the function of heterotrimeric kinesin II, block localization of Vg1 mRNA. We also show that exogenous Vg1 RNA or Xcat-2, another RNA that can use the late pathway, recruits endogenous kinesin II to the vegetal pole and colocalizes with it at the cortex. These data support a model in which kinesin II mediates the transport of specific RNA complexes destined for the vegetal cortex.
PubMed ID: 14761654
Article link: Curr Biol
Species referenced: Xenopus
Genes referenced: gdf1 gdf3 kif3a kif3b
Antibodies: Kif3b Ab1
Article Images: [+] show captions
|Figure 1. Expression and Localization of Heterotrimeric Kinesin II mRNA and Protein during Oogenesis(A–F) Whole mount in situ hybridization to endogenous Xklp3b (A–C) and Vg1 mRNA (D–F) in stage I (A and D), stage II (B and E), and stage III (C and F) oocytes. Arrows in (A)–(C) depict slight enrichment of Xklp3b mRNA in the mitochondrial cloud, wedge region, and vegetal cortex, respectively. GV is the germinal vesicle. The vegetal pole is oriented down in all images, and (C) and (F) are sectioned so that the cortex is visible. (G–I and K) Confocal images of Xklp3b-labeled Xenopus stage II (G), stage III (H), and stage IV (I) oocytes and an egg from Ciona intestinalis (K). Arrows in (H), (I), and (K) indicate enrichment of kinesin II at the vegetal cortex. (J) Western blots of whole-cell extracts prepared from stage I–VI oocytes via Xklp3a- and Xklp3b-specific antibodies. The scale bar represents 100 μm.|
|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).|
|Figure 3. Exogenous Vegetal RNA Recruits Endogenous Hetero-trimeric Kinesin II(A–C) Stage II oocytes were injected with low (5 fmol) or high (35 fmol) amounts of alexa fluor 546-labeled RNA (red), and immunocytochemistry was then performed with an anti-Xklp3b primary and Cy-2-labeled (green) secondary antibody for detection of kinesin II. Panels (A)–(C) show confocal images of oocytes injected with low amounts of the VgLE (A), high amounts of the VgLE (B), or high amounts of the Xcat-2 LE (C). The top image in all panels represents kinesin II, the middle panel shows the labeled RNA, and the bottom panel is an overlay of the two images. The RNA labels the wedge in all three oocytes (arrows), and recruitment of kinesin II to the wedge is clearly detected in oocytes injected with high amounts of the VgLE ([B] upper image) or Xcat-2 LE ([C] upper image). A low amount of the VgLE does not lead to detectable enrichment of kinesin II in the wedge (arrows) (A). (D) Higher magnification of the wedge and vegetal cortex from a late stage II oocyte shows colocalization (yellow) of the Xcat-2 LE and kinesin II most extensively in the vegetal cortex at the base of the wedge (arrowheads). The scale bar represents 100 μm in (A)–(C) and 10 μm in (D).|
|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).|
|kif3b (kinesin family member 3B) expressionin Xenopus laevis, assayed by in situ hybridization in oocyte stage I, vegetal view. (GV = germinal vesicle, arrow points to mitochondrial cloud).|
|gdf1 (growth differentiation factor 1) gene expressionin Xenopus laevis, assayed by in situ hybridization in oocyte stage II, vegetal pole down. (GV = germinal vesicle).|