Limits...
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

Identification of amino acid residues essential to the posterior localisation of Drosophila Vasa (DmVas) in the helicase superfamily C-terminal domain (HELICc).(A) Multiple sequence alignment of HELICc domains belonging to Vas orthologs of Drosophila melanogaster (Dm), the grasshopper Schistocerca gregaria (Sg), the pea aphid Acyrthosiphon pisum (Ap), and the mouse Mus musculus (Mm). Dark grey: conserved residues, Light grey: residues with similar properties. Residues substituted by Ala or Lys for the localisation assays shown in panels (B–O) are highlighted with red boxes. (B–O) Localisation analysis of green fluorescent protein (GFP)-tagged DmVas460–661 proteins with replaced amino acid residues in the HELICc sequence. Stage-10 egg chambers were stained using the anti-GFP antibody (green). Anterior is to the left and posterior is to the right. Scale bars, 25 μm. (B) DmVas460–661/S463A: replacement of the Ser463 with Ala is designated as S463A, and this applies to the other replacements. (C) DmVas460–661/S463K. (D) DmVas460–661/Y470A. (E) DmVas460–661/K480A. (F) DmVas460–661/483A. (G) DmVas460–661/E487A. (H) DmVas460–661/T498A. (I) DmVas460–661/L524A. (J) DmVas460–661/S526A. (K) DmVas460–661/Q527A. (L) DmVas460–661/H560A. (M) DmVas460–661/T590A. (N) DmVas460–661/P595A. (O) DmVas460–661/E596A. All of the previously described DmVas460–661 variants could be localised to the germ plasm, except DmVas460–661/Q527A, shown in panel (K). (P–R) Stage-5 egg chambers expressing GFP-DmVas460–661/Q527A were double stained using the anti-GFP (green) and anti-Krimp antibodies (red). (P) GFP-DmVas460–661/Q527A was not colocalised with (Q) Krimp in the nurse cells. Anterior is to the left and posterior is to the right. Scale bars, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4588571&req=5

f6: Identification of amino acid residues essential to the posterior localisation of Drosophila Vasa (DmVas) in the helicase superfamily C-terminal domain (HELICc).(A) Multiple sequence alignment of HELICc domains belonging to Vas orthologs of Drosophila melanogaster (Dm), the grasshopper Schistocerca gregaria (Sg), the pea aphid Acyrthosiphon pisum (Ap), and the mouse Mus musculus (Mm). Dark grey: conserved residues, Light grey: residues with similar properties. Residues substituted by Ala or Lys for the localisation assays shown in panels (B–O) are highlighted with red boxes. (B–O) Localisation analysis of green fluorescent protein (GFP)-tagged DmVas460–661 proteins with replaced amino acid residues in the HELICc sequence. Stage-10 egg chambers were stained using the anti-GFP antibody (green). Anterior is to the left and posterior is to the right. Scale bars, 25 μm. (B) DmVas460–661/S463A: replacement of the Ser463 with Ala is designated as S463A, and this applies to the other replacements. (C) DmVas460–661/S463K. (D) DmVas460–661/Y470A. (E) DmVas460–661/K480A. (F) DmVas460–661/483A. (G) DmVas460–661/E487A. (H) DmVas460–661/T498A. (I) DmVas460–661/L524A. (J) DmVas460–661/S526A. (K) DmVas460–661/Q527A. (L) DmVas460–661/H560A. (M) DmVas460–661/T590A. (N) DmVas460–661/P595A. (O) DmVas460–661/E596A. All of the previously described DmVas460–661 variants could be localised to the germ plasm, except DmVas460–661/Q527A, shown in panel (K). (P–R) Stage-5 egg chambers expressing GFP-DmVas460–661/Q527A were double stained using the anti-GFP (green) and anti-Krimp antibodies (red). (P) GFP-DmVas460–661/Q527A was not colocalised with (Q) Krimp in the nurse cells. Anterior is to the left and posterior is to the right. Scale bars, 20 μm.

Mentions: Posterior localisation of SgVasHELICc to the germ plasm in Drosophila (Fig. 3H–H”’) implies that the HELICc domains of SgVas and DmVas share common amino acids for interacting with Osk. To identify the amino acids critical for the germ plasm localisation of HELICc, we thus aligned the sequences from HELICc domains of DmVas, SgVas, and ApVas1 and selected 13 residues conserved in both DmVas and SgVas but not in ApVas1 (Fig. 6A). These include Ser463, an amino acid located within the first 10 amino acids (residues 460–469) of HELICc that have been demonstrated essential for germ plasm/nuage localisation and Osk interaction (Figs 3D–D”’,4D–D” and 5N). We then successively replaced all of the 13 target residues with Ala and monitored the localisation pattern of each HELICc with a single amino acid substitution. Our results showed that replacement of the Gln527 with Ala (Q527A) abolished the posterior localisation of HELICc (Fig. 6K). By contrast, restriction of HELICc to the germ plasm could still be visualized in the remaining substitutions including S463A (Fig. 6B,D–J,L–O). Further replacement of serine at position 463 with the basic amino acid lysine (S463K) did not impair HELICc localisation (Fig. 6C), indicating that Ser463 is not a key residue for germ plasm localisation of HELICc. In addition to germ plasm localisation, Q527A substitution abolished the nuage localisation of HELICc (Fig. 6P–R). To exclude the possibility that the absence of the posterior localisation of DmVas460–661/Q527A was caused by its weak expression or absence of expression, we compared the expression of DmVas460–661 and DmVas460–661/Q527A by performing western blot analysis and found that both proteins had similar expression levels in the ovary (Supplementary Fig. S3).


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)

Identification of amino acid residues essential to the posterior localisation of Drosophila Vasa (DmVas) in the helicase superfamily C-terminal domain (HELICc).(A) Multiple sequence alignment of HELICc domains belonging to Vas orthologs of Drosophila melanogaster (Dm), the grasshopper Schistocerca gregaria (Sg), the pea aphid Acyrthosiphon pisum (Ap), and the mouse Mus musculus (Mm). Dark grey: conserved residues, Light grey: residues with similar properties. Residues substituted by Ala or Lys for the localisation assays shown in panels (B–O) are highlighted with red boxes. (B–O) Localisation analysis of green fluorescent protein (GFP)-tagged DmVas460–661 proteins with replaced amino acid residues in the HELICc sequence. Stage-10 egg chambers were stained using the anti-GFP antibody (green). Anterior is to the left and posterior is to the right. Scale bars, 25 μm. (B) DmVas460–661/S463A: replacement of the Ser463 with Ala is designated as S463A, and this applies to the other replacements. (C) DmVas460–661/S463K. (D) DmVas460–661/Y470A. (E) DmVas460–661/K480A. (F) DmVas460–661/483A. (G) DmVas460–661/E487A. (H) DmVas460–661/T498A. (I) DmVas460–661/L524A. (J) DmVas460–661/S526A. (K) DmVas460–661/Q527A. (L) DmVas460–661/H560A. (M) DmVas460–661/T590A. (N) DmVas460–661/P595A. (O) DmVas460–661/E596A. All of the previously described DmVas460–661 variants could be localised to the germ plasm, except DmVas460–661/Q527A, shown in panel (K). (P–R) Stage-5 egg chambers expressing GFP-DmVas460–661/Q527A were double stained using the anti-GFP (green) and anti-Krimp antibodies (red). (P) GFP-DmVas460–661/Q527A was not colocalised with (Q) Krimp in the nurse cells. Anterior is to the left and posterior is to the right. Scale bars, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Identification of amino acid residues essential to the posterior localisation of Drosophila Vasa (DmVas) in the helicase superfamily C-terminal domain (HELICc).(A) Multiple sequence alignment of HELICc domains belonging to Vas orthologs of Drosophila melanogaster (Dm), the grasshopper Schistocerca gregaria (Sg), the pea aphid Acyrthosiphon pisum (Ap), and the mouse Mus musculus (Mm). Dark grey: conserved residues, Light grey: residues with similar properties. Residues substituted by Ala or Lys for the localisation assays shown in panels (B–O) are highlighted with red boxes. (B–O) Localisation analysis of green fluorescent protein (GFP)-tagged DmVas460–661 proteins with replaced amino acid residues in the HELICc sequence. Stage-10 egg chambers were stained using the anti-GFP antibody (green). Anterior is to the left and posterior is to the right. Scale bars, 25 μm. (B) DmVas460–661/S463A: replacement of the Ser463 with Ala is designated as S463A, and this applies to the other replacements. (C) DmVas460–661/S463K. (D) DmVas460–661/Y470A. (E) DmVas460–661/K480A. (F) DmVas460–661/483A. (G) DmVas460–661/E487A. (H) DmVas460–661/T498A. (I) DmVas460–661/L524A. (J) DmVas460–661/S526A. (K) DmVas460–661/Q527A. (L) DmVas460–661/H560A. (M) DmVas460–661/T590A. (N) DmVas460–661/P595A. (O) DmVas460–661/E596A. All of the previously described DmVas460–661 variants could be localised to the germ plasm, except DmVas460–661/Q527A, shown in panel (K). (P–R) Stage-5 egg chambers expressing GFP-DmVas460–661/Q527A were double stained using the anti-GFP (green) and anti-Krimp antibodies (red). (P) GFP-DmVas460–661/Q527A was not colocalised with (Q) Krimp in the nurse cells. Anterior is to the left and posterior is to the right. Scale bars, 20 μm.
Mentions: Posterior localisation of SgVasHELICc to the germ plasm in Drosophila (Fig. 3H–H”’) implies that the HELICc domains of SgVas and DmVas share common amino acids for interacting with Osk. To identify the amino acids critical for the germ plasm localisation of HELICc, we thus aligned the sequences from HELICc domains of DmVas, SgVas, and ApVas1 and selected 13 residues conserved in both DmVas and SgVas but not in ApVas1 (Fig. 6A). These include Ser463, an amino acid located within the first 10 amino acids (residues 460–469) of HELICc that have been demonstrated essential for germ plasm/nuage localisation and Osk interaction (Figs 3D–D”’,4D–D” and 5N). We then successively replaced all of the 13 target residues with Ala and monitored the localisation pattern of each HELICc with a single amino acid substitution. Our results showed that replacement of the Gln527 with Ala (Q527A) abolished the posterior localisation of HELICc (Fig. 6K). By contrast, restriction of HELICc to the germ plasm could still be visualized in the remaining substitutions including S463A (Fig. 6B,D–J,L–O). Further replacement of serine at position 463 with the basic amino acid lysine (S463K) did not impair HELICc localisation (Fig. 6C), indicating that Ser463 is not a key residue for germ plasm localisation of HELICc. In addition to germ plasm localisation, Q527A substitution abolished the nuage localisation of HELICc (Fig. 6P–R). To exclude the possibility that the absence of the posterior localisation of DmVas460–661/Q527A was caused by its weak expression or absence of expression, we compared the expression of DmVas460–661 and DmVas460–661/Q527A by performing western blot analysis and found that both proteins had similar expression levels in the ovary (Supplementary Fig. S3).

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