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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

Rescue vas mutant defects by Drosophila Vasa (DmVas) variants.Examination of abdominal segments and pole cells in embryos of wild-type or vas mutants expressing truncated or chimeric DmVas proteins. Genotypes: (A–B”) Wild-type: Oregon-R; (C–D”) vas mutant: vasPD/vasPH165; (E–F”) vas mutants expressing GFP-DmVas, (G–H”) GFP-DmVas158–661, (I–J”) GFP-DmVas220–661, (K–L”) GFP-ApD1 (ApVas11–60 + DmVas158–661), (M–N”) GFP-Ap90D (ApVas11–90 + DmVas158–661), and (O–P”) GFP-ApD2 (ApVas11–135 + DmVas220–661). (A,C,E,G,I,K,M,O) Cuticle preparations. Anterior is at the top. (A) Wild-type. (C) vasPD/vasPH165 embryo: no abdomen. (E,G,K,M) GFP-DmVas, GFP-DmVas158–661, GFP-ApD1, and GFP-Ap90D rescued abdominal defect. (I,O) GFP-DmVas220–661 and GFP-ApD2 did not rescue abdomen formation. (B–B”,D–D”,F–F”,H–H”,J–J”,L–L”,N–N”,P–P’) Z-stacks of confocal microscopic images of cellular blastoderm embryos double stained with anti-GFP and anti-Vas antibodies to visualise GFP-Vas variants (green) and endogenous Vas (red), respectively. Posterior is to the right. (B–B”) Wild-type. (D–D”) vasPD/vasPH165 embryo: no pole cell. (F–F”) GFP-DmVas rescued pole cell formation. (H–H”,L–L”) GFP-DmVas158–661 and GFP-ApD1 partially restored the pole cell number. (J–J”,P–P”) GFP-DmVas220–661 and GFP-ApD2 could not rescue pole cell formation. (N–N”) GFP-Ap90D accumulated in the posterior pole, but no pole cell was identified in most embryos examined.
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f7: Rescue vas mutant defects by Drosophila Vasa (DmVas) variants.Examination of abdominal segments and pole cells in embryos of wild-type or vas mutants expressing truncated or chimeric DmVas proteins. Genotypes: (A–B”) Wild-type: Oregon-R; (C–D”) vas mutant: vasPD/vasPH165; (E–F”) vas mutants expressing GFP-DmVas, (G–H”) GFP-DmVas158–661, (I–J”) GFP-DmVas220–661, (K–L”) GFP-ApD1 (ApVas11–60 + DmVas158–661), (M–N”) GFP-Ap90D (ApVas11–90 + DmVas158–661), and (O–P”) GFP-ApD2 (ApVas11–135 + DmVas220–661). (A,C,E,G,I,K,M,O) Cuticle preparations. Anterior is at the top. (A) Wild-type. (C) vasPD/vasPH165 embryo: no abdomen. (E,G,K,M) GFP-DmVas, GFP-DmVas158–661, GFP-ApD1, and GFP-Ap90D rescued abdominal defect. (I,O) GFP-DmVas220–661 and GFP-ApD2 did not rescue abdomen formation. (B–B”,D–D”,F–F”,H–H”,J–J”,L–L”,N–N”,P–P’) Z-stacks of confocal microscopic images of cellular blastoderm embryos double stained with anti-GFP and anti-Vas antibodies to visualise GFP-Vas variants (green) and endogenous Vas (red), respectively. Posterior is to the right. (B–B”) Wild-type. (D–D”) vasPD/vasPH165 embryo: no pole cell. (F–F”) GFP-DmVas rescued pole cell formation. (H–H”,L–L”) GFP-DmVas158–661 and GFP-ApD1 partially restored the pole cell number. (J–J”,P–P”) GFP-DmVas220–661 and GFP-ApD2 could not rescue pole cell formation. (N–N”) GFP-Ap90D accumulated in the posterior pole, but no pole cell was identified in most embryos examined.

Mentions: To explore the functions of the DmVas sequences N-terminal to the helicase core domain, we introduced transgenes encoding truncated DmVas proteins with various lengths of N-termini into the vas mutant (vasPD/vasPH165)79 and examined the recovered phenotypes. Compared with the wild-type embryos (Fig. 7A,B–B”), no abdominal segments or pole cells could be identified in the vas mutant embryos (Fig. 7C,D–D”). Expression of full-length DmVas in vas mutants recovered eight abdominal segments and 34.4 pole cells on average (Fig. 7E,F–F”; Supplementary Table S1), resembling the phenotypes observed in the wild-type embryos (Fig. 7A,B–B”). In the vas mutant embryos expressing DmVas158–661, a DmVas protein without the first 157 amino acids, we found that all the eight abdominal segments were formed (Fig. 7G; Supplementary Table S1) but only 20.6 pole cells, on average, were recovered (Fig. 7H–H”; Supplementary Table S1). However, when all the 219 amino acids N-terminal to the DEXDc domain were deleted (DmVas220–661; Fig. 7I,J–J”), the phenotype resembled that of the vas mutant (Fig. 7C,D–D”). We further analysed the ApVas1 N-terminal sequence to understand whether it could substitute the functions of the DmVas N-terminus. We expressed the chimeric proteins ApD1, Ap90D, or ApD2, all of whose N-termini were composed of various lengths of ApVas1 and found that the addition of the ApVas1 N-terminal sequence to DmVas158–661 (ApD1, Fig. 7L–L”; Ap90D, Fig. 7N–N”) or DmVas220–661 (ApD2, Fig. 7P–P”) did not enhance the rescue of pole cell formation defects in the vas mutant embryos. In comparison with ApD1, Ap90D expression (30-amino acid longer than ApD1) further hindered pole cell formation; practically, pole cells were not or barely formed (Fig. 7N–N”; Supplementary Table S1). By contrast, the expression of both ApD1 and Ap90D did not interfere with the rescue of abdominal segments (Fig. 7K,M). Nevertheless, when the sequence N-terminal to DEXDc of DmVas was replaced with the first 135 amino acids of ApVas1, its expression in the vas mutant embryos neither rescued the pole cells nor the abdominal segments (ApD2, Fig. 7O,P–P”).


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)

Rescue vas mutant defects by Drosophila Vasa (DmVas) variants.Examination of abdominal segments and pole cells in embryos of wild-type or vas mutants expressing truncated or chimeric DmVas proteins. Genotypes: (A–B”) Wild-type: Oregon-R; (C–D”) vas mutant: vasPD/vasPH165; (E–F”) vas mutants expressing GFP-DmVas, (G–H”) GFP-DmVas158–661, (I–J”) GFP-DmVas220–661, (K–L”) GFP-ApD1 (ApVas11–60 + DmVas158–661), (M–N”) GFP-Ap90D (ApVas11–90 + DmVas158–661), and (O–P”) GFP-ApD2 (ApVas11–135 + DmVas220–661). (A,C,E,G,I,K,M,O) Cuticle preparations. Anterior is at the top. (A) Wild-type. (C) vasPD/vasPH165 embryo: no abdomen. (E,G,K,M) GFP-DmVas, GFP-DmVas158–661, GFP-ApD1, and GFP-Ap90D rescued abdominal defect. (I,O) GFP-DmVas220–661 and GFP-ApD2 did not rescue abdomen formation. (B–B”,D–D”,F–F”,H–H”,J–J”,L–L”,N–N”,P–P’) Z-stacks of confocal microscopic images of cellular blastoderm embryos double stained with anti-GFP and anti-Vas antibodies to visualise GFP-Vas variants (green) and endogenous Vas (red), respectively. Posterior is to the right. (B–B”) Wild-type. (D–D”) vasPD/vasPH165 embryo: no pole cell. (F–F”) GFP-DmVas rescued pole cell formation. (H–H”,L–L”) GFP-DmVas158–661 and GFP-ApD1 partially restored the pole cell number. (J–J”,P–P”) GFP-DmVas220–661 and GFP-ApD2 could not rescue pole cell formation. (N–N”) GFP-Ap90D accumulated in the posterior pole, but no pole cell was identified in most embryos examined.
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f7: Rescue vas mutant defects by Drosophila Vasa (DmVas) variants.Examination of abdominal segments and pole cells in embryos of wild-type or vas mutants expressing truncated or chimeric DmVas proteins. Genotypes: (A–B”) Wild-type: Oregon-R; (C–D”) vas mutant: vasPD/vasPH165; (E–F”) vas mutants expressing GFP-DmVas, (G–H”) GFP-DmVas158–661, (I–J”) GFP-DmVas220–661, (K–L”) GFP-ApD1 (ApVas11–60 + DmVas158–661), (M–N”) GFP-Ap90D (ApVas11–90 + DmVas158–661), and (O–P”) GFP-ApD2 (ApVas11–135 + DmVas220–661). (A,C,E,G,I,K,M,O) Cuticle preparations. Anterior is at the top. (A) Wild-type. (C) vasPD/vasPH165 embryo: no abdomen. (E,G,K,M) GFP-DmVas, GFP-DmVas158–661, GFP-ApD1, and GFP-Ap90D rescued abdominal defect. (I,O) GFP-DmVas220–661 and GFP-ApD2 did not rescue abdomen formation. (B–B”,D–D”,F–F”,H–H”,J–J”,L–L”,N–N”,P–P’) Z-stacks of confocal microscopic images of cellular blastoderm embryos double stained with anti-GFP and anti-Vas antibodies to visualise GFP-Vas variants (green) and endogenous Vas (red), respectively. Posterior is to the right. (B–B”) Wild-type. (D–D”) vasPD/vasPH165 embryo: no pole cell. (F–F”) GFP-DmVas rescued pole cell formation. (H–H”,L–L”) GFP-DmVas158–661 and GFP-ApD1 partially restored the pole cell number. (J–J”,P–P”) GFP-DmVas220–661 and GFP-ApD2 could not rescue pole cell formation. (N–N”) GFP-Ap90D accumulated in the posterior pole, but no pole cell was identified in most embryos examined.
Mentions: To explore the functions of the DmVas sequences N-terminal to the helicase core domain, we introduced transgenes encoding truncated DmVas proteins with various lengths of N-termini into the vas mutant (vasPD/vasPH165)79 and examined the recovered phenotypes. Compared with the wild-type embryos (Fig. 7A,B–B”), no abdominal segments or pole cells could be identified in the vas mutant embryos (Fig. 7C,D–D”). Expression of full-length DmVas in vas mutants recovered eight abdominal segments and 34.4 pole cells on average (Fig. 7E,F–F”; Supplementary Table S1), resembling the phenotypes observed in the wild-type embryos (Fig. 7A,B–B”). In the vas mutant embryos expressing DmVas158–661, a DmVas protein without the first 157 amino acids, we found that all the eight abdominal segments were formed (Fig. 7G; Supplementary Table S1) but only 20.6 pole cells, on average, were recovered (Fig. 7H–H”; Supplementary Table S1). However, when all the 219 amino acids N-terminal to the DEXDc domain were deleted (DmVas220–661; Fig. 7I,J–J”), the phenotype resembled that of the vas mutant (Fig. 7C,D–D”). We further analysed the ApVas1 N-terminal sequence to understand whether it could substitute the functions of the DmVas N-terminus. We expressed the chimeric proteins ApD1, Ap90D, or ApD2, all of whose N-termini were composed of various lengths of ApVas1 and found that the addition of the ApVas1 N-terminal sequence to DmVas158–661 (ApD1, Fig. 7L–L”; Ap90D, Fig. 7N–N”) or DmVas220–661 (ApD2, Fig. 7P–P”) did not enhance the rescue of pole cell formation defects in the vas mutant embryos. In comparison with ApD1, Ap90D expression (30-amino acid longer than ApD1) further hindered pole cell formation; practically, pole cells were not or barely formed (Fig. 7N–N”; Supplementary Table S1). By contrast, the expression of both ApD1 and Ap90D did not interfere with the rescue of abdominal segments (Fig. 7K,M). Nevertheless, when the sequence N-terminal to DEXDc of DmVas was replaced with the first 135 amino acids of ApVas1, its expression in the vas mutant embryos neither rescued the pole cells nor the abdominal segments (ApD2, Fig. 7O,P–P”).

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