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In vivo mapping of the functional regions of the DEAD-box helicase Vasa.

Dehghani M, Lasko P - Biol Open (2015)

Bottom Line: We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification.One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases.Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, 3649 Promenade Sir William Osler, Montréal, QC H3G 0B1, Canada.

No MeSH data available.


Localization of eGFP-Vas proteins in vas1 ovaries.(A) N-terminal deletions. (B) Mutations in conserved DEAD-box helicase motifs. (C) C-terminal mutations. For each genotype the left, the top right and the bottom right images show a stage 5 egg chamber (confocal image), an early stage 10 egg chamber, and a stage 14 oocyte. Scale bars (50 µm) are included on the top set of images; all corresponding images from other genotypes are at the same magnification. (D) Higher magnification confocal images comparing distribution of eGFP-Vas at the posterior pole of vas1; egfp-vas+ and vas1;egfp-vasΔ636-646 stage 14 oocytes. eGFP-VasΔ636-646 distribution is more diffuse. For each genotype the top image shows the middle focal plane whereas the bottom image illustrates the maximum intensity projection of the z stack. Scale bars = 50 µm.
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f04: Localization of eGFP-Vas proteins in vas1 ovaries.(A) N-terminal deletions. (B) Mutations in conserved DEAD-box helicase motifs. (C) C-terminal mutations. For each genotype the left, the top right and the bottom right images show a stage 5 egg chamber (confocal image), an early stage 10 egg chamber, and a stage 14 oocyte. Scale bars (50 µm) are included on the top set of images; all corresponding images from other genotypes are at the same magnification. (D) Higher magnification confocal images comparing distribution of eGFP-Vas at the posterior pole of vas1; egfp-vas+ and vas1;egfp-vasΔ636-646 stage 14 oocytes. eGFP-VasΔ636-646 distribution is more diffuse. For each genotype the top image shows the middle focal plane whereas the bottom image illustrates the maximum intensity projection of the z stack. Scale bars = 50 µm.

Mentions: First, we examined the spatial distribution of each mutant form of eGFP-Vas. In early oogenesis, wild-type Vas accumulates in the perinuclear nuage of the nurse cells. Beginning at stage 10, Vas is then transported into the oocyte where it accumulates in the posterior pole plasm. This localization pattern is recapitulated by transgenically expressed wild-type eGFP-Vas, and by eGFP-Vas deleted for amino acids 3-139 or for several smaller segments within that interval (Fig. 4A). However, a more extensive N-terminal deletion of amino acids 3-200 made the association of Vas with the perinuclear nuage weaker and reduced Vas localization to the pole plasm (Fig. 4A).


In vivo mapping of the functional regions of the DEAD-box helicase Vasa.

Dehghani M, Lasko P - Biol Open (2015)

Localization of eGFP-Vas proteins in vas1 ovaries.(A) N-terminal deletions. (B) Mutations in conserved DEAD-box helicase motifs. (C) C-terminal mutations. For each genotype the left, the top right and the bottom right images show a stage 5 egg chamber (confocal image), an early stage 10 egg chamber, and a stage 14 oocyte. Scale bars (50 µm) are included on the top set of images; all corresponding images from other genotypes are at the same magnification. (D) Higher magnification confocal images comparing distribution of eGFP-Vas at the posterior pole of vas1; egfp-vas+ and vas1;egfp-vasΔ636-646 stage 14 oocytes. eGFP-VasΔ636-646 distribution is more diffuse. For each genotype the top image shows the middle focal plane whereas the bottom image illustrates the maximum intensity projection of the z stack. Scale bars = 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400588&req=5

f04: Localization of eGFP-Vas proteins in vas1 ovaries.(A) N-terminal deletions. (B) Mutations in conserved DEAD-box helicase motifs. (C) C-terminal mutations. For each genotype the left, the top right and the bottom right images show a stage 5 egg chamber (confocal image), an early stage 10 egg chamber, and a stage 14 oocyte. Scale bars (50 µm) are included on the top set of images; all corresponding images from other genotypes are at the same magnification. (D) Higher magnification confocal images comparing distribution of eGFP-Vas at the posterior pole of vas1; egfp-vas+ and vas1;egfp-vasΔ636-646 stage 14 oocytes. eGFP-VasΔ636-646 distribution is more diffuse. For each genotype the top image shows the middle focal plane whereas the bottom image illustrates the maximum intensity projection of the z stack. Scale bars = 50 µm.
Mentions: First, we examined the spatial distribution of each mutant form of eGFP-Vas. In early oogenesis, wild-type Vas accumulates in the perinuclear nuage of the nurse cells. Beginning at stage 10, Vas is then transported into the oocyte where it accumulates in the posterior pole plasm. This localization pattern is recapitulated by transgenically expressed wild-type eGFP-Vas, and by eGFP-Vas deleted for amino acids 3-139 or for several smaller segments within that interval (Fig. 4A). However, a more extensive N-terminal deletion of amino acids 3-200 made the association of Vas with the perinuclear nuage weaker and reduced Vas localization to the pole plasm (Fig. 4A).

Bottom Line: We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification.One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases.Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, 3649 Promenade Sir William Osler, Montréal, QC H3G 0B1, Canada.

No MeSH data available.