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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.


Time course of pole cell development in eGFP-Vas+ and eGFP-VasΔ636-646 expressing embryos.(A) A series of still shots from live imaging of pole cell formation in eGFP-Vas+ expressing embryos. eGFP-Vas is tightly localized at the posterior pole and then concentrates in foci within pole buds. Posterior nuclear divisions become asynchronous with somatic nuclear divisions, and eGFP-Vas positive pole cells completely form. (B) A series of still shots from live imaging of pole cell formation in eGFP-VasΔ636-646 expressing embryos. eGFP-Vas is less tightly localized at the posterior pole and forms more foci than in wild type. Mitosis remains synchronous throughout the embryo. eGFP-Vas is then lost from the posterior region and pole cells fail to form. Scale bar = 50 µm.
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f07: Time course of pole cell development in eGFP-Vas+ and eGFP-VasΔ636-646 expressing embryos.(A) A series of still shots from live imaging of pole cell formation in eGFP-Vas+ expressing embryos. eGFP-Vas is tightly localized at the posterior pole and then concentrates in foci within pole buds. Posterior nuclear divisions become asynchronous with somatic nuclear divisions, and eGFP-Vas positive pole cells completely form. (B) A series of still shots from live imaging of pole cell formation in eGFP-VasΔ636-646 expressing embryos. eGFP-Vas is less tightly localized at the posterior pole and forms more foci than in wild type. Mitosis remains synchronous throughout the embryo. eGFP-Vas is then lost from the posterior region and pole cells fail to form. Scale bar = 50 µm.

Mentions: One C-terminal mutant, eGFP-VasΔ636-646, when expressed at levels comparable to the wild-type control, restored almost no pole cell formation activity to vas1 embryos (Fig. 6). When examined in more detail, however, its phenotype is novel in that pole buds begin to form but then regress. Unlike in wild-type, mitotic divisions remain synchronous in the somatic and posterior pole region of embryos expressing eGFP-VasΔ636-646 (Fig. 7; supplementary material Movies 1, 2). We hypothesize that the more diffuse localization of eGFP-VasΔ636-646 at the posterior region results in an insufficient concentration of Vas at the extreme posterior to catalyze pole cell formation, and as mitosis remains synchronous, the eGFP-Vas that is present gets divided into a larger number of foci than in wild-type. Supporting this hypothesis, higher expression of eGFP-VasΔ636-646 enables many more vas1 embryos to produce pole cells, however the number of pole cells each embryo produces is highly variable, and on average is much lower than that for example in vas1; egfp-vasΔ15-75, which is expressed at a comparable level (4.3±0.3 versus 9.9±0.2) (supplementary material Fig. S4). We did not observe a similar phenotype for eGFP-VasR644A, a point mutation that affects a potential arginine methylation site within the 636-646 interval.


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

Dehghani M, Lasko P - Biol Open (2015)

Time course of pole cell development in eGFP-Vas+ and eGFP-VasΔ636-646 expressing embryos.(A) A series of still shots from live imaging of pole cell formation in eGFP-Vas+ expressing embryos. eGFP-Vas is tightly localized at the posterior pole and then concentrates in foci within pole buds. Posterior nuclear divisions become asynchronous with somatic nuclear divisions, and eGFP-Vas positive pole cells completely form. (B) A series of still shots from live imaging of pole cell formation in eGFP-VasΔ636-646 expressing embryos. eGFP-Vas is less tightly localized at the posterior pole and forms more foci than in wild type. Mitosis remains synchronous throughout the embryo. eGFP-Vas is then lost from the posterior region and pole cells fail to form. Scale bar = 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f07: Time course of pole cell development in eGFP-Vas+ and eGFP-VasΔ636-646 expressing embryos.(A) A series of still shots from live imaging of pole cell formation in eGFP-Vas+ expressing embryos. eGFP-Vas is tightly localized at the posterior pole and then concentrates in foci within pole buds. Posterior nuclear divisions become asynchronous with somatic nuclear divisions, and eGFP-Vas positive pole cells completely form. (B) A series of still shots from live imaging of pole cell formation in eGFP-VasΔ636-646 expressing embryos. eGFP-Vas is less tightly localized at the posterior pole and forms more foci than in wild type. Mitosis remains synchronous throughout the embryo. eGFP-Vas is then lost from the posterior region and pole cells fail to form. Scale bar = 50 µm.
Mentions: One C-terminal mutant, eGFP-VasΔ636-646, when expressed at levels comparable to the wild-type control, restored almost no pole cell formation activity to vas1 embryos (Fig. 6). When examined in more detail, however, its phenotype is novel in that pole buds begin to form but then regress. Unlike in wild-type, mitotic divisions remain synchronous in the somatic and posterior pole region of embryos expressing eGFP-VasΔ636-646 (Fig. 7; supplementary material Movies 1, 2). We hypothesize that the more diffuse localization of eGFP-VasΔ636-646 at the posterior region results in an insufficient concentration of Vas at the extreme posterior to catalyze pole cell formation, and as mitosis remains synchronous, the eGFP-Vas that is present gets divided into a larger number of foci than in wild-type. Supporting this hypothesis, higher expression of eGFP-VasΔ636-646 enables many more vas1 embryos to produce pole cells, however the number of pole cells each embryo produces is highly variable, and on average is much lower than that for example in vas1; egfp-vasΔ15-75, which is expressed at a comparable level (4.3±0.3 versus 9.9±0.2) (supplementary material Fig. S4). We did not observe a similar phenotype for eGFP-VasR644A, a point mutation that affects a potential arginine methylation site within the 636-646 interval.

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.