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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 of chimeric proteins composed of Drosophila Vasa (DmVas) and pea aphid Vasa (ApVas1) in the oocyte.(A) Schematic illustration of domain swapping between DmVas and ApVas1. Blue and pink colours represent sequences derived from DmVas and ApVas1, respectively. The same colour codes are used in the other Fig. of this paper. (B–H’”) Localisation analysis of the green fluorescent protein (GFP)-tagged chimeric proteins in the oocytes during oogenesis from stages 9–13. (B–B”’) Posterior localisation of GFP-DmVas: a positive control. (C–C”’) GFP-ApVas1: posterior localisation was not detected. (D–D”’) GFP-DAp1 (DmVas1–157 + ApVas161–579; Osk interacting motif (OIM) was not included): posterior localisation was not detected. (E–E”’) GFP-DAp2 (DmVas1–320 + ApVas1241–579; OIM was included): posterior localisation was not detected. (F–F”’) GFP-ApD1 (ApVas11–60 + DmVas158–661; OIM was included): posterior localisation was detected. (G–G”’) GFP-ApD2 (ApVas11–135 + DmVas220–661; OIM was partially truncated): posterior localisation was detected. (H–H”’) GFP-ApD3 (ApVas11–240 + DmVas321–661; OIM was not included): posterior localisation became prominent from Stage 10a onwards. In all panels, anterior is to the left and posterior is to the right. Scale bars, 25 μm.
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f2: Localisation of chimeric proteins composed of Drosophila Vasa (DmVas) and pea aphid Vasa (ApVas1) in the oocyte.(A) Schematic illustration of domain swapping between DmVas and ApVas1. Blue and pink colours represent sequences derived from DmVas and ApVas1, respectively. The same colour codes are used in the other Fig. of this paper. (B–H’”) Localisation analysis of the green fluorescent protein (GFP)-tagged chimeric proteins in the oocytes during oogenesis from stages 9–13. (B–B”’) Posterior localisation of GFP-DmVas: a positive control. (C–C”’) GFP-ApVas1: posterior localisation was not detected. (D–D”’) GFP-DAp1 (DmVas1–157 + ApVas161–579; Osk interacting motif (OIM) was not included): posterior localisation was not detected. (E–E”’) GFP-DAp2 (DmVas1–320 + ApVas1241–579; OIM was included): posterior localisation was not detected. (F–F”’) GFP-ApD1 (ApVas11–60 + DmVas158–661; OIM was included): posterior localisation was detected. (G–G”’) GFP-ApD2 (ApVas11–135 + DmVas220–661; OIM was partially truncated): posterior localisation was detected. (H–H”’) GFP-ApD3 (ApVas11–240 + DmVas321–661; OIM was not included): posterior localisation became prominent from Stage 10a onwards. In all panels, anterior is to the left and posterior is to the right. Scale bars, 25 μm.

Mentions: In Drosophila, the germ plasm localisation of Vas is dependent on the pre-deposition of Osk in the posterior pole of the oocyte78. By using yeast two-hybrid and GST pull-down assays, the DmVas sequences required for the Osk–Vas interaction had been found to span amino acids 163–3193132; here we refer to this region as the Osk interaction motif (OIM). Such physical interaction between Osk and DmVas has been considered essential for the germ plasm localisation of DmVas. In order to determine whether the lack of OIM sequence is responsible for the failure of posterior localisation of ApVas1 in Drosophila oocyte, we performed domain-swapping analyses replacing various lengths of N-terminal ApVas1 sequences with N-terminal DmVas sequences (Fig. 2A). DAp1 is a chimeric protein formed by replacing the first 60 amino acids of ApVas1 with the N-terminal 157 amino acids of DmVas (Fig. 2A). As expected, unlike DmVas (Fig. 2B–B”’), DAp1 was not localised to the posterior germ plasm (Fig. 2D–D”’) the same as ApVas1 (Fig. 2C–C”’). However, to our surprise, DAp2 whose sequence contained an intact OIM in the N-terminal 320 residues of DmVas (DmVas1–320) was still not posteriorly localised to the oocyte (Fig. 2E–E”’).


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 of chimeric proteins composed of Drosophila Vasa (DmVas) and pea aphid Vasa (ApVas1) in the oocyte.(A) Schematic illustration of domain swapping between DmVas and ApVas1. Blue and pink colours represent sequences derived from DmVas and ApVas1, respectively. The same colour codes are used in the other Fig. of this paper. (B–H’”) Localisation analysis of the green fluorescent protein (GFP)-tagged chimeric proteins in the oocytes during oogenesis from stages 9–13. (B–B”’) Posterior localisation of GFP-DmVas: a positive control. (C–C”’) GFP-ApVas1: posterior localisation was not detected. (D–D”’) GFP-DAp1 (DmVas1–157 + ApVas161–579; Osk interacting motif (OIM) was not included): posterior localisation was not detected. (E–E”’) GFP-DAp2 (DmVas1–320 + ApVas1241–579; OIM was included): posterior localisation was not detected. (F–F”’) GFP-ApD1 (ApVas11–60 + DmVas158–661; OIM was included): posterior localisation was detected. (G–G”’) GFP-ApD2 (ApVas11–135 + DmVas220–661; OIM was partially truncated): posterior localisation was detected. (H–H”’) GFP-ApD3 (ApVas11–240 + DmVas321–661; OIM was not included): posterior localisation became prominent from Stage 10a onwards. In all panels, anterior is to the left and posterior is to the right. Scale bars, 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Localisation of chimeric proteins composed of Drosophila Vasa (DmVas) and pea aphid Vasa (ApVas1) in the oocyte.(A) Schematic illustration of domain swapping between DmVas and ApVas1. Blue and pink colours represent sequences derived from DmVas and ApVas1, respectively. The same colour codes are used in the other Fig. of this paper. (B–H’”) Localisation analysis of the green fluorescent protein (GFP)-tagged chimeric proteins in the oocytes during oogenesis from stages 9–13. (B–B”’) Posterior localisation of GFP-DmVas: a positive control. (C–C”’) GFP-ApVas1: posterior localisation was not detected. (D–D”’) GFP-DAp1 (DmVas1–157 + ApVas161–579; Osk interacting motif (OIM) was not included): posterior localisation was not detected. (E–E”’) GFP-DAp2 (DmVas1–320 + ApVas1241–579; OIM was included): posterior localisation was not detected. (F–F”’) GFP-ApD1 (ApVas11–60 + DmVas158–661; OIM was included): posterior localisation was detected. (G–G”’) GFP-ApD2 (ApVas11–135 + DmVas220–661; OIM was partially truncated): posterior localisation was detected. (H–H”’) GFP-ApD3 (ApVas11–240 + DmVas321–661; OIM was not included): posterior localisation became prominent from Stage 10a onwards. In all panels, anterior is to the left and posterior is to the right. Scale bars, 25 μm.
Mentions: In Drosophila, the germ plasm localisation of Vas is dependent on the pre-deposition of Osk in the posterior pole of the oocyte78. By using yeast two-hybrid and GST pull-down assays, the DmVas sequences required for the Osk–Vas interaction had been found to span amino acids 163–3193132; here we refer to this region as the Osk interaction motif (OIM). Such physical interaction between Osk and DmVas has been considered essential for the germ plasm localisation of DmVas. In order to determine whether the lack of OIM sequence is responsible for the failure of posterior localisation of ApVas1 in Drosophila oocyte, we performed domain-swapping analyses replacing various lengths of N-terminal ApVas1 sequences with N-terminal DmVas sequences (Fig. 2A). DAp1 is a chimeric protein formed by replacing the first 60 amino acids of ApVas1 with the N-terminal 157 amino acids of DmVas (Fig. 2A). As expected, unlike DmVas (Fig. 2B–B”’), DAp1 was not localised to the posterior germ plasm (Fig. 2D–D”’) the same as ApVas1 (Fig. 2C–C”’). However, to our surprise, DAp2 whose sequence contained an intact OIM in the N-terminal 320 residues of DmVas (DmVas1–320) was still not posteriorly localised to the oocyte (Fig. 2E–E”’).

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