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Germ plasm localisation of the HELICc of Vasa in Drosophila: analysis of domain sufficiency and amino acids critical for localisation.

Wang SC, Hsu HJ, Lin GW, Wang TF, Chang CC, Lin MD - Sci Rep (2015)

Bottom Line: We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation.We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species.This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien, Taiwan.

ABSTRACT
Formation of the germ plasm drives germline specification in Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

No MeSH data available.


Related in: MedlinePlus

Oskar (Osk) interacts with the helicase superfamily C-terminal domain (HELICc) of Drosophila Vasa (DmVas) in vivo and in vitro.(A–L) Localisation analyses of (A–D) green fluorescent protein (GFP)-DmVas, (E–H) GFP-Vas460–621/HELICc, and (I–L) GFP-SgVasHELICc were performed in egg chambers at Stage 10 by immunostaining with the anti-GFP antibody. Genetic backgrounds: (A,E,I) Wild-type; (B,F,J) osk mutant with the genotype osk54/Df(3R)pXT103; (C,G,K) vls  mutant with the genotype Df(2L)Pr2b,P[barren+]/Df(2L)be408; (D,H,L) tud mutant with the genotype tudtux46/Df(2R)PF1. All the 3 GFP-tagged Vas proteins could be localised to the posterior germ plasm, except in the osk mutant background. (M,N) Yeast two-hybrid analysis performed using the β-galactosidase colony lift filter assay. (M) None of the singly transformed ‘bait’ and ‘prey’ plasmids could induce the expression of the lacZ reporter. (N) Osk could interact with full-length DmVas and DmVas460–621/HELICc. (O–T) Stage-10 egg chambers were stained with the anti-GFP (green) and anti-Osk antibodies (red). (Q,T) Merged images. (O–Q) GFP-DmVas460–621/HELICc was colocalised with Osk in the germ plasm. (R–T) In the egg chambers coexpressing the GFP-DmVas460–621/HELICc and osk-bcd 3′UTR transcripts, the GFP-DmVas460–621/HELICc was colocalised with Osk in the anterior and posterior poles of the oocyte. In the panels with egg chambers (A–L,O–T), anterior is to the left and posterior is to the right. The DmVas460–621/HELICc is abbreviated as DmVas460–621 in all the panels. Scale bars, 25 μm.
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f5: Oskar (Osk) interacts with the helicase superfamily C-terminal domain (HELICc) of Drosophila Vasa (DmVas) in vivo and in vitro.(A–L) Localisation analyses of (A–D) green fluorescent protein (GFP)-DmVas, (E–H) GFP-Vas460–621/HELICc, and (I–L) GFP-SgVasHELICc were performed in egg chambers at Stage 10 by immunostaining with the anti-GFP antibody. Genetic backgrounds: (A,E,I) Wild-type; (B,F,J) osk mutant with the genotype osk54/Df(3R)pXT103; (C,G,K) vls mutant with the genotype Df(2L)Pr2b,P[barren+]/Df(2L)be408; (D,H,L) tud mutant with the genotype tudtux46/Df(2R)PF1. All the 3 GFP-tagged Vas proteins could be localised to the posterior germ plasm, except in the osk mutant background. (M,N) Yeast two-hybrid analysis performed using the β-galactosidase colony lift filter assay. (M) None of the singly transformed ‘bait’ and ‘prey’ plasmids could induce the expression of the lacZ reporter. (N) Osk could interact with full-length DmVas and DmVas460–621/HELICc. (O–T) Stage-10 egg chambers were stained with the anti-GFP (green) and anti-Osk antibodies (red). (Q,T) Merged images. (O–Q) GFP-DmVas460–621/HELICc was colocalised with Osk in the germ plasm. (R–T) In the egg chambers coexpressing the GFP-DmVas460–621/HELICc and osk-bcd 3′UTR transcripts, the GFP-DmVas460–621/HELICc was colocalised with Osk in the anterior and posterior poles of the oocyte. In the panels with egg chambers (A–L,O–T), anterior is to the left and posterior is to the right. The DmVas460–621/HELICc is abbreviated as DmVas460–621 in all the panels. Scale bars, 25 μm.

Mentions: To address how HELICc was localised to germ plasm, we investigated whether its posterior localisation in the oocyte was dependent on Osk. In wild-type egg chambers, both the full-length DmVas (Fig. 5A) and the sole HELICc (DmVas460–621/HELICc, Fig. 5E) were localised to the germ plasm. Similar to full-length DmVas as previously reported37 (Fig. 5B), the posterior localisation of HELICc was not observed in the osk mutant egg chambers (Fig. 5F). As both Valois (Vls)38 and Tudor (Tud)39 are two known downstream components of Osk, we further tested whether Vls and Tud can assist the posterior localisation of HELICc and found that the posterior localisation of both full-length DmVas (Fig. 5C,D) and HELICc (Fig. 5G,H) were not affect in either vls or tud mutant backgrounds. Ectopic expression of SgVasHELICc in Drosophila showed that SgVasHELICc could also be posteriorly localised (Fig. 5I), and its localisation patterns resembled those of the HELICc of DmVas in various genetic backgrounds (Fig. 5J–L). These results suggest that the germ plasm localisation of HELICc was dependent on Osk but not Vls or Tud.


Germ plasm localisation of the HELICc of Vasa in Drosophila: analysis of domain sufficiency and amino acids critical for localisation.

Wang SC, Hsu HJ, Lin GW, Wang TF, Chang CC, Lin MD - Sci Rep (2015)

Oskar (Osk) interacts with the helicase superfamily C-terminal domain (HELICc) of Drosophila Vasa (DmVas) in vivo and in vitro.(A–L) Localisation analyses of (A–D) green fluorescent protein (GFP)-DmVas, (E–H) GFP-Vas460–621/HELICc, and (I–L) GFP-SgVasHELICc were performed in egg chambers at Stage 10 by immunostaining with the anti-GFP antibody. Genetic backgrounds: (A,E,I) Wild-type; (B,F,J) osk mutant with the genotype osk54/Df(3R)pXT103; (C,G,K) vls  mutant with the genotype Df(2L)Pr2b,P[barren+]/Df(2L)be408; (D,H,L) tud mutant with the genotype tudtux46/Df(2R)PF1. All the 3 GFP-tagged Vas proteins could be localised to the posterior germ plasm, except in the osk mutant background. (M,N) Yeast two-hybrid analysis performed using the β-galactosidase colony lift filter assay. (M) None of the singly transformed ‘bait’ and ‘prey’ plasmids could induce the expression of the lacZ reporter. (N) Osk could interact with full-length DmVas and DmVas460–621/HELICc. (O–T) Stage-10 egg chambers were stained with the anti-GFP (green) and anti-Osk antibodies (red). (Q,T) Merged images. (O–Q) GFP-DmVas460–621/HELICc was colocalised with Osk in the germ plasm. (R–T) In the egg chambers coexpressing the GFP-DmVas460–621/HELICc and osk-bcd 3′UTR transcripts, the GFP-DmVas460–621/HELICc was colocalised with Osk in the anterior and posterior poles of the oocyte. In the panels with egg chambers (A–L,O–T), anterior is to the left and posterior is to the right. The DmVas460–621/HELICc is abbreviated as DmVas460–621 in all the panels. Scale bars, 25 μm.
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Related In: Results  -  Collection

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f5: Oskar (Osk) interacts with the helicase superfamily C-terminal domain (HELICc) of Drosophila Vasa (DmVas) in vivo and in vitro.(A–L) Localisation analyses of (A–D) green fluorescent protein (GFP)-DmVas, (E–H) GFP-Vas460–621/HELICc, and (I–L) GFP-SgVasHELICc were performed in egg chambers at Stage 10 by immunostaining with the anti-GFP antibody. Genetic backgrounds: (A,E,I) Wild-type; (B,F,J) osk mutant with the genotype osk54/Df(3R)pXT103; (C,G,K) vls mutant with the genotype Df(2L)Pr2b,P[barren+]/Df(2L)be408; (D,H,L) tud mutant with the genotype tudtux46/Df(2R)PF1. All the 3 GFP-tagged Vas proteins could be localised to the posterior germ plasm, except in the osk mutant background. (M,N) Yeast two-hybrid analysis performed using the β-galactosidase colony lift filter assay. (M) None of the singly transformed ‘bait’ and ‘prey’ plasmids could induce the expression of the lacZ reporter. (N) Osk could interact with full-length DmVas and DmVas460–621/HELICc. (O–T) Stage-10 egg chambers were stained with the anti-GFP (green) and anti-Osk antibodies (red). (Q,T) Merged images. (O–Q) GFP-DmVas460–621/HELICc was colocalised with Osk in the germ plasm. (R–T) In the egg chambers coexpressing the GFP-DmVas460–621/HELICc and osk-bcd 3′UTR transcripts, the GFP-DmVas460–621/HELICc was colocalised with Osk in the anterior and posterior poles of the oocyte. In the panels with egg chambers (A–L,O–T), anterior is to the left and posterior is to the right. The DmVas460–621/HELICc is abbreviated as DmVas460–621 in all the panels. Scale bars, 25 μm.
Mentions: To address how HELICc was localised to germ plasm, we investigated whether its posterior localisation in the oocyte was dependent on Osk. In wild-type egg chambers, both the full-length DmVas (Fig. 5A) and the sole HELICc (DmVas460–621/HELICc, Fig. 5E) were localised to the germ plasm. Similar to full-length DmVas as previously reported37 (Fig. 5B), the posterior localisation of HELICc was not observed in the osk mutant egg chambers (Fig. 5F). As both Valois (Vls)38 and Tudor (Tud)39 are two known downstream components of Osk, we further tested whether Vls and Tud can assist the posterior localisation of HELICc and found that the posterior localisation of both full-length DmVas (Fig. 5C,D) and HELICc (Fig. 5G,H) were not affect in either vls or tud mutant backgrounds. Ectopic expression of SgVasHELICc in Drosophila showed that SgVasHELICc could also be posteriorly localised (Fig. 5I), and its localisation patterns resembled those of the HELICc of DmVas in various genetic backgrounds (Fig. 5J–L). These results suggest that the germ plasm localisation of HELICc was dependent on Osk but not Vls or Tud.

Bottom Line: We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation.We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species.This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien, Taiwan.

ABSTRACT
Formation of the germ plasm drives germline specification in Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

No MeSH data available.


Related in: MedlinePlus