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Rab11 polarization of the Drosophila oocyte: a novel link between membrane trafficking, microtubule organization, and oskar mRNA localization and translation

Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA

View larger version (33K): [in a new window]   Fig. 1. rab11 cloning and genetics. (A) Organization of the rab11 locus. The top line shows the 6.3 kb genomic rescuing fragment, the position of the P element insertion and the transcription start site and direction (arrow). The structure of the rabex1 and rabex2 excision alleles is shown below the rescue fragment, where the broken lines indicate uncertainties in the locations of the breakpoints. The rab11 transcription unit is shown at the bottom. Exons are depicted as rectangles, with the filled regions corresponding to protein coding segments. A, Asp718I; X, XhoI. (B) Developmental Northern blot showing rab11 expression throughout the fly life cycle. The control blot in the bottom panel was probed for Adh mRNA. RNA was prepared from the following tissues and stages: Ov, adult female ovaries; e, 0-24 h embryos; L1-L3, first, second and third instar larvae, respectively; m, adult males; f, adult females. (C) In situ hybridization for osk mRNA in rab11P2148 and rabex1 GLCs. The absence of osk transcripts in rabex1 GLCs indicates that an oocyte is not determined. Similar results were obtained with rabex2 GLCs. (D) Western Blot with Rab11 antisera. Equivalent amounts of total ovarian protein from wild-type (WT) and rab11P2148/rab+ flies (rab/+) were applied to each lane. A single major band of the expected size for Rab11 is detected in each lane.

View larger version (97K): [in a new window]   Fig. 2. Rab11 expression in wild-type oocytes and in rab11P2148 GLCs. (A-E) Confocal immunolocalization of Rab11 and Osk. (A-C) wild-type oocytes. (A) Stage 7 oocyte showing strong accumulation of Rab11 along the lateral and posterior cortex, and in a perinuclear compartment (arrowhead). In these and in all subsequent panels, anterior is towards the left. (B) Stage 10 oocyte showing specific accumulation of Rab11 at the posterior pole. (C) Early stage 10 oocyte doubly labeled for Rab11 (red) and Osk (green). The two proteins show near perfect colocalization at the posterior pole as evident by the mixed (yellow) fluorescent signal. (D-E) Confocal immunolocalization of Rab11 in rab11P2148 GLCs. (D) Late stage 7 oocyte showing normal accumulation of Rab11 in perinuclear compartment and along the cell cortex. (E) Early stage 10 oocyte showing diffuse accumulation of Rab11 throughout ooplasm with slight enrichment along cell cortex. (F) Rab11-GFP expression in a living wild-type stage 10 oocyte.

View larger version (78K): [in a new window]   Fig. 3. Transferrin recycling in wild-type oocyte and in rab11P2148 GLCs. (A,B) A wild-type stage 9 oocyte cultured with Texas Red-transferrin for one minute followed by a 20 minute wash in media alone. (A) Red channel showing the accumulation of transferrin in the perivitelline compartment and in vesicles at posterior pole of the oocyte. (B) Merged image showing transferrin (red) and Rab11-GFP (green). Inset shows an enlarged view of posterior pole, where the accumulation of transferrin in intracellular vesicles is clearly evident. (C) rab11P2148 GLC cultured with transferrin as described above. Most of the transferrin is in the ooplasm, not in the perivitelline compartment. (D-G) FITC-Lectin labeling of plasma and vitelline membranes. (D-E) Plasma membrane labeling with Lycopersicon esculentum lectin. (F-G) Vitelline membrane labeling with Datura stramonium lectin. (D,F) Wild type. (E,G) rab11P2148 GLCs.

View larger version (78K): [in a new window]   Fig. 4. Rab11-dependent localization of -adaptin to the posterior pole of the oocyte. (A-C) Wild-type stage 9 oocyte doubly labeled for Rab11 (red) and -adaptin (green). (C) Merged image. (D) Merged image of a stage 10 oocyte doubly labeled for Rab11 (red) and -adaptin (green). In both oocytes, -adaptin is abundant all along the plasma membrane and in a posterior compartment that is close to, but clearly distinct from, the Rab11 expression domain. (E-G) A late stage9/early stage 10 rab11P2148 GLC doubly labeled for Rab11 (red) and -adaptin (green), where the merged image is shown in G. Neither protein is found at high levels at the posterior pole of the oocyte.

View larger version (105K): [in a new window]   Fig. 5. The localization of Rab11 and -adaptin to the posterior pole of the oocyte is reinforced by Osk, but does not require a polarized microtubule cytoskeleton. Confocal immunolocalization of Rab11 (red) and -adaptin in stage 9 osk protein null (A-C) and wild-type (D) oocytes. Rab11 is localized to the posterior pole of osk protein null oocytes (A), but at a reduced level compared with that in wild type (see text). Normal or near normal amounts of -adaptin accumulate at the posterior pole of osk protein null oocytes (B), but the protein is distinctly more cortical than in wild-type oocytes. Note, for example, that while -adaptin (upward pointing arrows) is anterior to Rab11 (downward pointing arrows) in wild-type oocytes (D), it is posterior to Rab11 in the osk protein null oocyte (C). (E-I) Confocal immunolocalization of Rab11 (red) and Stau (green) in grk null oocytes. (E-G) Stage 9 oocyte showing specific accumulation of Rab11 at the posterior pole of the oocyte (E) and Stauffen protein at the cell center and posterior pole (F). (G) Merged image. (H,I) A rare stage 10 grk null oocyte in which Stauffen protein is found exclusively or almost exclusively at the cell center (I), consistent with complete depolarization of the microtubule cytoskeleton of the oocyte . Even though no microtubule polarity is evident in this oocyte, Rab11 accumulates specifically at the posterior pole of the oocyte (H). Although it is difficult to rule out the Rab11 signal in H is from neighboring (posterior) follicle cells, this possibility seems unlikely given that no specific accumulation of Rab11 is seen in posterior follicle cells of other grk nulls (see E). Moreover, anterior and posterior follicle cells are thought to adopt identical (i.e. anterior) fates in grk mutants (Roth et al., 1995; Gonzäles-Reyes et al., 1995). The -adaptin expression pattern in grk null oocytes resembles that seen in the osk protein null oocytes (data not shown).

View larger version (113K): [in a new window]   Fig. 6. Microtubule organization in wild-type oocytes and in rab11P2148 GLCs. (A-F) Organization of microtubule minus ends in wild-type oocytes and in rab11P2148 GLCs. Expression of tau-GFP in living stage 7 oocytes from wild-type (B) and rab11P2148 GLC-bearing flies (E). Intense fluorescence at anterior cortex and the absence of fluorescence at the posterior pole indicate normal reorganization of the microtubule cytoskeleton in both oocytes, resulting in a concentration of microtubule minus ends at the anterior cortex. Immunolocalization of -tubulin in wild-type stage 8 oocytes (A) and in stage 8 rab11P2148 GLCs (D). A distinct anterior-posterior gradient of microtubule density is seen in both oocytes, again indicative of normal microtubule minus end organization. In situ hybridization for bicoid (C) and gurken (F) transcripts in stage 10 rab11P2148 GLCs. Both patterns are indistinguishable from that seen in wild-type controls (Saunders and Cohen, 1999; St Johnston, 1995). (G-I) Organization of microtubule plus ends in wild-type oocytes and in rab11P2148 GLCs. Immunolocalization of Kin:ß-gal fusion protein in wild-type stage 9 oocytes (G) and in early (H) and late (I) stage 9 rab11P2148 GLCs. The tight fluorescence at the posterior tip of the wild-type oocyte indicates sharp focusing of microtubule plus ends. Conversely, the expanded fluorescence along the lateral and posterior cortexes of rab11P2148 GLCs indicates poor focusing of microtubule plus ends.

View larger version (96K): [in a new window]   Fig. 7. Rab11 is required for osk mRNA transport to the posterior pole and for its subsequent anchoring and translation. (A-C) The distribution of osk mRNA (A,B) and protein (C) in wild-type oocytes. Osk mRNA is transported to the posterior end of the oocyte during stage 8 and forms a ball-shaped complex (A). During stage 9, the ball resolved into a cap-shaped structure (B) that persists through the completion of oogenesis. Osk (C) is first detected coincident with the osk mRNA ball to cap transition. (D-G) The distribution of osk mRNA (D,E,G) and protein (F) in rab11P2148 GLCs. osk mRNA is slow to accumulate at the posterior pole of rab11P2148 GLCs. A mass of osk mRNA is often seen at the center of stage 8 rab11P2148 GLCs (D), possibly reflecting stalled transport. Ultimately, osk mRNA reaches the posterior pole of rab11P2148 GLCs, but the mRNA remains as a ball (E), or often breaks up into several smaller balls (G). Consistent with a defect in anchoring, the osk mRNA is not translated as evident by the absence of detectable Osk (F).

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