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First published online 13 February 2008
doi: 10.1242/dev.017293

Development 135, 1107-1117 (2008)
Published by The Company of Biologists 2008
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The endocytic pathway acts downstream of Oskar in Drosophila germ plasm assembly

Tsubasa Tanaka and Akira Nakamura*

Laboratory for Germline Development, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan.


Figure 1
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  		Fig. 1. Isolation of Drosophila Rabenosyn-5 homolog. (A,B) GFP-Vas localization in wild-type (A) and C241 GLC (B) oocytes. Enlarged images of the bracketed regions are shown in the right panels. Mutant clones were identified by the lack of nuclear GFP signals in the germline. (C) Domain structure of CG8506 and its orthologs. The positions of the C2H2 and FYVE (Fab1p, YOTB, Vac1p and EEA1) zinc fingers, coiled-coil domain and NPF (Asn-Pro-Phe) motif are indicated. The C241 mutant contains a premature stop codon at the residue 315 (asterisk). (D) Western blot of ovarian extracts from wild-type and C241 heterozygotes with rabbit and rat polyclonal anti-Rbsn-5 antisera. A single band of the expected size for Rbsn-5 was detected. A truncated protein resulting from a premature stop codon (expected size of 35 kD; asterisk) was undetectable in C241/+ lysates. (E) Drosophila Rbsn-5 specifically interacts with the GTP-bound form of Rab5. In-vitro-translated Rbsn-5 was incubated with GST-Rab5, GST-Rab11 and GST-Rab7 in the presence of GDP or GTP{gamma}S. Bound proteins were analyzed by western blot using anti-Rbsn-5 antisera. (F,G) Auto-fluorescent granules derived from endocytosed yolk were detected in wild-type oocytes (F) but not in rbsn-5- oocytes (G). (H,I) Incorporation of the FM4-64 dye by wild-type (H) and rbsn-5- (I) oocytes. No internalization of FM4-64 dye was detected in rbsn-5- oocytes. Anterior is to the left, posterior is to the right in all images. Scale bars: 20 µm.

 

Figure 2
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  		Fig. 2. Rbsn-5 is required for osk RNP localization and the maintenance of MT polarity. (A,C,E) Stau localization in stage 6-10 Drosophila oocytes of wild type (A), rbsn-5- GLC (C) and rbsn-5- GLC expressing the rbsn-5 transgene (E). Arrowheads point to mislocalized Stau signals in rbsn-5- oocytes. F-actin was counterstained with Alexa-660-conjugated phalloidin (cyan). (B,D,F) Osk localization in stage 10 oocytes of wild type (B), rbsn-5- GLC (D) and rbsn-5- GLC expressing the rbsn-5 transgene (F). DNA stained with DAPI is shown in cyan. (G-J) In situ hybridization for osk (green) and grk or bcd (magenta) RNAs in wild-type (G,H) or rbsn-5- GLC (I,J) stage 9 oocytes. DNA stained with DAPI is shown in grayscale. In rbsn-5- oocytes, osk RNA localization was disrupted (arrows), but grk or bcd RNA localization was unaffected (arrowheads). (K-N) Stau and Kin-βgal distribution in stage 8-9 wild-type (K,L) or rbsn-5- GLC (M,N) oocytes. Arrowheads in N indicate aberrantly localized Stau and Kin-βgal in the rbsn-5- GLCs. DNA was counterstained with DAPI (cyan). Scale bars: 20 µm.

 

Figure 3
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  		Fig. 3. Polarized distribution of Rbsn-5 and endosomal proteins in developing Drosophila oocytes. (A,B) Double staining of wild-type oocytes for Rbsn-5 (green) and Stau (magenta). Both Rbsn-5 and Stau were concentrated at the center of stage 7 oocytes (A, arrowhead). From stage 8 onward, both signals were restricted to the posterior pole of the oocytes (B). An enlarged image of the bracketed region is shown in the right panel. (C-H) Distribution of endosomal proteins (Rab5, Rab11 and Rab7) in wild-type oocytes. All endosomal proteins showed transient accumulation at the center of stage 7 oocytes and posterior enrichment in late-stage oocytes (arrowheads). (I-L) Double staining of wild-type ovaries for Stau (magenta) and ER (I,J) or Golgi apparatus (K,L) markers (green). DNA was counterstained with DAPI (cyan) in all images. Scale bars: 20 µm.

 

Figure 4
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  		Fig. 4. Polarized endocytic activity within the Drosophila oocyte depends on Osk and Grk. (A-C) Distribution of Stau, Rbsn-5, Rab11 and incorporated FM4-64 dye, as indicated, in wild-type (A), osk54/Df(3R)pXT103 (B), and grkHK36/Df(2L)ED623 (C) oocytes at stages 8 and 10. DNA in all images was visualized by DAPI staining (cyan). Note that signals for Rbsn-5 on lateral cortex were consistently reduced in osk mutant oocytes at stage 8 (B), and that Rab11 failed to localize to the center of grk mutant oocytes (C). We presently do not know why these differences occur. (D-F) Percentages of oocytes showing a posterior accumulation of Rbsn-5 and Rab11 in wild-type (D), osk54/Df(3R)pXT103 (E) and grkHK36/Df(2L)ED623 (F) oocytes during stages 8-10. Error bars denote the standard deviation from at least three independent experiments. Scale bars: 20 µm.

 

Figure 5
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  		Fig. 5. Anterior Osk, specifically long Osk, recruits endosomal proteins and stimulates endocytosis in Drosophila. (A-D) Rbsn-5 localization at the oocyte anterior without (A) or with expression of osk-bcd 3' UTR (B), osk(M1L)-bcd 3' UTR (C) or osk(M139L)-bcd 3' UTR (D). Rbsn-5 accumulated in the anterior of oocytes expressing the intact osk-bcd 3' UTR or osk(M139L)-bcd 3' UTR transgenes, but not the osk(M1L)-bcd 3' UTR transgene (arrowheads). (E-J) Rab5 (E,F), Rab11 (G,H) and Rab7 (I,J) localization at the oocyte anterior without (E,G,I) or with long Osk expression (F,H,J). Rab5, Rab11 and Rab7 all accumulated at the oocyte anterior when long Osk was expressed (arrowheads). (K,L) Incorporation of FM4-64 in stage 9 oocytes without (K) or with long Osk expression (L). Ectopic dye incorporation was observed at the oocyte anterior in the presence of long Osk (arrowheads). (M,N) Kin-βgal localization without (M) or with long Osk expression (N). No ectopic Kin-βgal signal was detected at the anterior pole of the osk(M139L)-bcd 3' UTR oocyte (arrowheads). DNA was counterstained with DAPI (cyan) in all images. Scale bars: 20 µm.

 

Figure 6
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  		Fig. 6. Rbsn-5 is required for Osk-mediated F-actin reorganization and the anchoring of pole plasm components in Drosophila. (A-D) Wild-type (A,B) or rbsn-5- (C,D) oocytes, without (A,C) or with expression of osk-bcd 3' UTR (B,D), were stained for Osk and F-actin. Enlargements of the bracketed regions are shown in the right panels. Aberrant F-actin aggregates (magenta) that contained Osk (green) were observed around the anterior region in the rbsn-5- oocyte when the osk-bcd 3' UTR transgene was expressed (arrowheads in D). (E-H) Tud protein localization in wild-type (E,F) or rbsn-5- (G,H) oocytes without (E,G) or with expression of osk-bcd 3' UTR (F,H). Arrowheads in H indicate aberrant aggregates containing F-actin and Tud. (I-N) Rab5 (I,J), Rab11 (K,L) and Rab7 (M,N) localization to the oocyte anterior without (I,K,M) or with expression of osk-bcd 3' UTR (J,L,N) in rbsn-5- GLCs. Arrowheads point to the accumulation of endosomal proteins in areas of ectopic Osk expression. DNA was counterstained with DAPI (cyan). Scale bars: 20 µm.

 

Figure 7
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  		Fig. 7. Model for the roles of the endocytic pathway in pole plasm assembly. The polarization of the oocyte by the Grk signal promotes the rearrangement of the MT array and initial accumulation of endosomal proteins to the oocyte posterior (arrow 1). The endocytic activity maintains MT polarity (arrow 2), which is required for osk RNA localization to the oocyte posterior. Long Osk, translated from the localized osk message, recruits endosomal proteins and upregulates endocytic activity at the oocyte posterior (arrow 3). The stimulated endocytic pathway in turn regulates the F-actin reorganization that is essential to anchor the pole plasm components to the oocyte cortex (arrow 4). The close association of endosomal proteins with osk RNP in developing oocytes (see Fig. 3) suggests that the endocytic pathway might also play a role in the targeting of osk RNP to the oocyte posterior (the arrow with a broken line).

 

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