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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

Localisation analysis of truncated Drosophila Vasa (DmVas) proteins, chimeric Vas proteins, and helicase superfamily C-terminal domains (HELICc) from other animal species.All Vas proteins were green fluorescent protein (GFP)-tagged and analyses were performed in egg chambers from Stages 9–13 of oogenesis. Each panel shows the posterior half of the oocyte. (A–A”’) GFP-DmVas460–661: sequence N-terminal to HELICc was truncated; (B–B”’) GFP-DmVas460–621/HELICc (abbreviated as DmVas460–621): a sole HELICc. Posterior localisation of DmVas460–661 and DmVas460–621/HELICc was detected; (C–C”’) GFP-DmVas1–460: a C-terminal truncated DmVas polypeptide without the HELICc and C-terminal sequences. (D–D”’) GFP-DmVas470–661: an N-terminal truncated DmVas polypeptide without the N-terminal sequence, DEXDc, and 10 residues in the N-terminus of HELICc. Posterior localisation of DmVas1–460 and DmVas470–661 was not detected; (E–E”’) GFP-ApDHELICc: a ApVas1–DmVas chimeric protein in which the N-terminal sequence and most of the DEXDc domain sequences of DmVas were replaced by those from ApVas1. Posterior localisation was detected; (F–F”’ to I–I”’) GFP-tagged HELICc of Vas orthologs from the pea aphid (F–F”’, GFP-ApVas1HELICc), cricket (G–G”’, GFP-GbVasHELICc), grasshopper (H–H”’, GFP-SgVasHELICc), and mouse (I–I”’, GFP-MvhHELICc). Posterior localisation could be detected only in the egg chamber expressing SgVasHELICc (H–H”’). In all panels, anterior is to the left and posterior is to the right. Scale bars, 25 μm.
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f3: Localisation analysis of truncated Drosophila Vasa (DmVas) proteins, chimeric Vas proteins, and helicase superfamily C-terminal domains (HELICc) from other animal species.All Vas proteins were green fluorescent protein (GFP)-tagged and analyses were performed in egg chambers from Stages 9–13 of oogenesis. Each panel shows the posterior half of the oocyte. (A–A”’) GFP-DmVas460–661: sequence N-terminal to HELICc was truncated; (B–B”’) GFP-DmVas460–621/HELICc (abbreviated as DmVas460–621): a sole HELICc. Posterior localisation of DmVas460–661 and DmVas460–621/HELICc was detected; (C–C”’) GFP-DmVas1–460: a C-terminal truncated DmVas polypeptide without the HELICc and C-terminal sequences. (D–D”’) GFP-DmVas470–661: an N-terminal truncated DmVas polypeptide without the N-terminal sequence, DEXDc, and 10 residues in the N-terminus of HELICc. Posterior localisation of DmVas1–460 and DmVas470–661 was not detected; (E–E”’) GFP-ApDHELICc: a ApVas1–DmVas chimeric protein in which the N-terminal sequence and most of the DEXDc domain sequences of DmVas were replaced by those from ApVas1. Posterior localisation was detected; (F–F”’ to I–I”’) GFP-tagged HELICc of Vas orthologs from the pea aphid (F–F”’, GFP-ApVas1HELICc), cricket (G–G”’, GFP-GbVasHELICc), grasshopper (H–H”’, GFP-SgVasHELICc), and mouse (I–I”’, GFP-MvhHELICc). Posterior localisation could be detected only in the egg chamber expressing SgVasHELICc (H–H”’). In all panels, anterior is to the left and posterior is to the right. Scale bars, 25 μm.

Mentions: Given that ApD3 without an OIM could still be localised to the germ plasm (Fig. 2H’–H”’), we inferred that sequences within DEXDc or HELICc may contribute to the posterior localisation of DmVas. To test this inference, we further truncated all the amino acids N-terminal to HELICc and found that DmVas could still be localised to the germ plasm from stage 10 onwards (DmVas460–661, Fig. 3A–A”’). Similar patterns could also be observed in DmVas containing a single HELICc (DmVas460–621/HELICc, Fig. 3B–B”’). By contrast, a DmVas protein whose HELICc and sequences C-terminal to HELICc were all truncated was not posteriorly localised (DmVas1–460, Fig. 3C–C”’). We further found that deletion of the residues 460–469 in HELICc disabled the posterior localisation (DmVas470–661, Fig. 3D–D”’). When sequences located N-terminal to HELICc were replaced with corresponding ApVas1 sequences, posterior localisation still occurred (ApDHELICc, Fig. 3E–E”’). Altogether, these results suggest that residues 460–469 within HELICc are critical for the posterior localisation of DmVas to the germ plasm. However, we did not observe germ plasm localisation of HELICc derived from ApVas1 (ApVas1HELICc, Fig. 3F–F”’), cricket Gryllus bimaculatus Vasa (GbVasHELICc, Fig. 3G–G”’), or the mouse Vas homolog protein (MvhHELICc, Fig. 3I–I”’) in the Drosophila oocytes. These HELICc proteins, instead, were uniformly distributed in the cytoplasm. Although a low level of localisation of the GbVasHELICc was identified in the cortex of the oocyte, it was not particularly enriched to the germ plasm (Fig. 3G’”). The HELICc of grasshopper Schistocerca gregaria Vasa (SgVasHELICc), surprisingly, was localised to the germ plasm of Drosophila oocyte (Fig. 3H–H”’), yet in S. gregaria a maternal germ plasm expressing SgVas had not been identified33. In addition to the germ plasm, DmVas is known to be localised to the nuage, an electron-dense structure restricted to the nuclear periphery of nurse cells8233435. Here, we used Krimp as a nuage marker to investigate whether truncated DmVas proteins could be localised to the nuage36. We found that DmVas460–661 (Fig. 4B–B”) and DmVas460–621/HELICc (Fig. 4C–C”), both of which containing intact HELICc, could be colocalised with Krimp to the nuage as full length DmVas (Fig. 4A–A”). By contrast, the shortened HELICc lacking the residues 460–469 was mis-localised to the nucleus and did not exhibit nuage localisation in the perinuclear region (DmVas470–661, Fig. 4D–D”). Similar to DmVas, HELICc of SgVas was restricted to the nuage and colocalised with Krimp (SgVasHELICc, Fig. 4E–E”). These results suggest that the HELICc domain could also encompass amino acids critical for nuage localisation.


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)

Localisation analysis of truncated Drosophila Vasa (DmVas) proteins, chimeric Vas proteins, and helicase superfamily C-terminal domains (HELICc) from other animal species.All Vas proteins were green fluorescent protein (GFP)-tagged and analyses were performed in egg chambers from Stages 9–13 of oogenesis. Each panel shows the posterior half of the oocyte. (A–A”’) GFP-DmVas460–661: sequence N-terminal to HELICc was truncated; (B–B”’) GFP-DmVas460–621/HELICc (abbreviated as DmVas460–621): a sole HELICc. Posterior localisation of DmVas460–661 and DmVas460–621/HELICc was detected; (C–C”’) GFP-DmVas1–460: a C-terminal truncated DmVas polypeptide without the HELICc and C-terminal sequences. (D–D”’) GFP-DmVas470–661: an N-terminal truncated DmVas polypeptide without the N-terminal sequence, DEXDc, and 10 residues in the N-terminus of HELICc. Posterior localisation of DmVas1–460 and DmVas470–661 was not detected; (E–E”’) GFP-ApDHELICc: a ApVas1–DmVas chimeric protein in which the N-terminal sequence and most of the DEXDc domain sequences of DmVas were replaced by those from ApVas1. Posterior localisation was detected; (F–F”’ to I–I”’) GFP-tagged HELICc of Vas orthologs from the pea aphid (F–F”’, GFP-ApVas1HELICc), cricket (G–G”’, GFP-GbVasHELICc), grasshopper (H–H”’, GFP-SgVasHELICc), and mouse (I–I”’, GFP-MvhHELICc). Posterior localisation could be detected only in the egg chamber expressing SgVasHELICc (H–H”’). In all panels, anterior is to the left and posterior is to the right. Scale bars, 25 μm.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4588571&req=5

f3: Localisation analysis of truncated Drosophila Vasa (DmVas) proteins, chimeric Vas proteins, and helicase superfamily C-terminal domains (HELICc) from other animal species.All Vas proteins were green fluorescent protein (GFP)-tagged and analyses were performed in egg chambers from Stages 9–13 of oogenesis. Each panel shows the posterior half of the oocyte. (A–A”’) GFP-DmVas460–661: sequence N-terminal to HELICc was truncated; (B–B”’) GFP-DmVas460–621/HELICc (abbreviated as DmVas460–621): a sole HELICc. Posterior localisation of DmVas460–661 and DmVas460–621/HELICc was detected; (C–C”’) GFP-DmVas1–460: a C-terminal truncated DmVas polypeptide without the HELICc and C-terminal sequences. (D–D”’) GFP-DmVas470–661: an N-terminal truncated DmVas polypeptide without the N-terminal sequence, DEXDc, and 10 residues in the N-terminus of HELICc. Posterior localisation of DmVas1–460 and DmVas470–661 was not detected; (E–E”’) GFP-ApDHELICc: a ApVas1–DmVas chimeric protein in which the N-terminal sequence and most of the DEXDc domain sequences of DmVas were replaced by those from ApVas1. Posterior localisation was detected; (F–F”’ to I–I”’) GFP-tagged HELICc of Vas orthologs from the pea aphid (F–F”’, GFP-ApVas1HELICc), cricket (G–G”’, GFP-GbVasHELICc), grasshopper (H–H”’, GFP-SgVasHELICc), and mouse (I–I”’, GFP-MvhHELICc). Posterior localisation could be detected only in the egg chamber expressing SgVasHELICc (H–H”’). In all panels, anterior is to the left and posterior is to the right. Scale bars, 25 μm.
Mentions: Given that ApD3 without an OIM could still be localised to the germ plasm (Fig. 2H’–H”’), we inferred that sequences within DEXDc or HELICc may contribute to the posterior localisation of DmVas. To test this inference, we further truncated all the amino acids N-terminal to HELICc and found that DmVas could still be localised to the germ plasm from stage 10 onwards (DmVas460–661, Fig. 3A–A”’). Similar patterns could also be observed in DmVas containing a single HELICc (DmVas460–621/HELICc, Fig. 3B–B”’). By contrast, a DmVas protein whose HELICc and sequences C-terminal to HELICc were all truncated was not posteriorly localised (DmVas1–460, Fig. 3C–C”’). We further found that deletion of the residues 460–469 in HELICc disabled the posterior localisation (DmVas470–661, Fig. 3D–D”’). When sequences located N-terminal to HELICc were replaced with corresponding ApVas1 sequences, posterior localisation still occurred (ApDHELICc, Fig. 3E–E”’). Altogether, these results suggest that residues 460–469 within HELICc are critical for the posterior localisation of DmVas to the germ plasm. However, we did not observe germ plasm localisation of HELICc derived from ApVas1 (ApVas1HELICc, Fig. 3F–F”’), cricket Gryllus bimaculatus Vasa (GbVasHELICc, Fig. 3G–G”’), or the mouse Vas homolog protein (MvhHELICc, Fig. 3I–I”’) in the Drosophila oocytes. These HELICc proteins, instead, were uniformly distributed in the cytoplasm. Although a low level of localisation of the GbVasHELICc was identified in the cortex of the oocyte, it was not particularly enriched to the germ plasm (Fig. 3G’”). The HELICc of grasshopper Schistocerca gregaria Vasa (SgVasHELICc), surprisingly, was localised to the germ plasm of Drosophila oocyte (Fig. 3H–H”’), yet in S. gregaria a maternal germ plasm expressing SgVas had not been identified33. In addition to the germ plasm, DmVas is known to be localised to the nuage, an electron-dense structure restricted to the nuclear periphery of nurse cells8233435. Here, we used Krimp as a nuage marker to investigate whether truncated DmVas proteins could be localised to the nuage36. We found that DmVas460–661 (Fig. 4B–B”) and DmVas460–621/HELICc (Fig. 4C–C”), both of which containing intact HELICc, could be colocalised with Krimp to the nuage as full length DmVas (Fig. 4A–A”). By contrast, the shortened HELICc lacking the residues 460–469 was mis-localised to the nucleus and did not exhibit nuage localisation in the perinuclear region (DmVas470–661, Fig. 4D–D”). Similar to DmVas, HELICc of SgVas was restricted to the nuage and colocalised with Krimp (SgVasHELICc, Fig. 4E–E”). These results suggest that the HELICc domain could also encompass amino acids critical for nuage localisation.

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