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Btk29A-mediated tyrosine phosphorylation of armadillo/β-catenin promotes ring canal growth in Drosophila oogenesis.

Hamada-Kawaguchi N, Nishida Y, Yamamoto D - PLoS ONE (2015)

Bottom Line: In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29AficP mutation.This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm) are located.We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology and Neurosciences, Tohoku University, Graduate School of Life Sciences, Sendai, Japan.

ABSTRACT
Drosophila Btk29A is the ortholog of mammalian Btk, a Tec family nonreceptor tyrosine kinase whose deficit causes X-linked agammaglobulinemia in humans. The Btk29AficP mutation induces multiple abnormalities in oogenesis, including the growth arrest of ring canals, large intercellular bridges that allow the flow of cytoplasm carrying maternal products essential for embryonic development from the nurse cells to the oocyte during oogenesis. In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29AficP mutation. This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm) are located. Our previous in vitro and in vivo analyses revealed that Btk29A directly phosphorylates Arm, leading to its release from DE-cadherin. In the present experiments, immunohistological analysis revealed that phosphorylation at tyrosine 150 (Y150) and Y667 of Arm was diminished in Btk29AficP mutant ring canals. Overexpression of an Arm mutant with unphosphorylatable Y150 inhibited ring canal growth. Thus Btk29A-induced Y150 phosphorylation is necessary for the normal growth of ring canals. We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.

No MeSH data available.


Related in: MedlinePlus

Effect of the Btk29AficP mutation on the localization of Btk29A, Arm and phosphotyrosine.The panels show the stage 2 (A, B, E and F) and the stage 1 (C, D, G and H) egg chambers from wild-type (A—D) and Btk29AficP mutant (E—H) ovaries doubly stained with phalloidin for actin (red in A, C, E and G) and the anti-Btk29A antibody (green in A and E) or the anti-phosphotyrosine antibody (green in C and G). The images of antibody staining are also shown in black and white in (B), (D), (F) and (H) to aid in comparisons of the localization and abundance of Btk29A or phosphotyrosine between the wild-type and Btk29AficP mutant egg chambers. Btk29A is highly enriched around ring canals and cell-cell contact regions in the wild-type chambers, whereas it is barely detectable in the Btk29AficP mutant chambers. (I—N) Arm (green) is localized along the cell-cell contact regions and ring canals in the wild-type (I) and Btk29AficP mutant (K) germaria. Actin is visualized by phalloidin staining (red). The entire stage 1 (J and L) and close-up views of ring canals in stage 1 (M and N) of the wild-type (J and M) and Btk29AficP mutant (L and N) germaria are shown. Some of the ring canals are marked with arrowheads in (A—L). Note the marked accumulations of Arm in the regions surrounding the ring canals in Btk29AficP mutants. Scale bars: 10 μm.
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pone.0121484.g003: Effect of the Btk29AficP mutation on the localization of Btk29A, Arm and phosphotyrosine.The panels show the stage 2 (A, B, E and F) and the stage 1 (C, D, G and H) egg chambers from wild-type (A—D) and Btk29AficP mutant (E—H) ovaries doubly stained with phalloidin for actin (red in A, C, E and G) and the anti-Btk29A antibody (green in A and E) or the anti-phosphotyrosine antibody (green in C and G). The images of antibody staining are also shown in black and white in (B), (D), (F) and (H) to aid in comparisons of the localization and abundance of Btk29A or phosphotyrosine between the wild-type and Btk29AficP mutant egg chambers. Btk29A is highly enriched around ring canals and cell-cell contact regions in the wild-type chambers, whereas it is barely detectable in the Btk29AficP mutant chambers. (I—N) Arm (green) is localized along the cell-cell contact regions and ring canals in the wild-type (I) and Btk29AficP mutant (K) germaria. Actin is visualized by phalloidin staining (red). The entire stage 1 (J and L) and close-up views of ring canals in stage 1 (M and N) of the wild-type (J and M) and Btk29AficP mutant (L and N) germaria are shown. Some of the ring canals are marked with arrowheads in (A—L). Note the marked accumulations of Arm in the regions surrounding the ring canals in Btk29AficP mutants. Scale bars: 10 μm.

Mentions: It has been reported that germline clones for strong mutant alleles in the Btk29A locus display several distinct phenotypes, such as ring canal undergrowth [15, 17], fusome distortion [14], defects in karyosome formation [14], packaging defects [14], aberrant border cell migration (N. Hamada-Kawaguchi, unpublished data) and oocyte mislocalization [15]. In keeping with these observations, Btk29AficP mutant ovaries, expressing only the short type 1 splice form of Btk29A, were grossly aberrant as a result of the irregular shapes and sizes of germ cells and follicle cells (Figs. 1B, 1D and 1F). Moreover, the Btk29AficP egg chambers were equipped with ring canals that were much smaller than those of the wild type (Figs. 2A, 2B, 2E and 2F): mature wild-type egg chambers have ring canals of 8–10 μm in diameter (Fig. 2I), whereas those of Btk29AficP were about 3–5 μm in diameter (Fig. 2I), as reported for other Btk29A alleles that affect both type 1 and type 2 products ([15, 17] and N. Hamada-Kawaguchi, unpublished data). It is notable that the lumina of mutant ring canals appeared to be very narrow in Btk29AficP mutants, compared to the wild type (Figs. 3M and 3N).


Btk29A-mediated tyrosine phosphorylation of armadillo/β-catenin promotes ring canal growth in Drosophila oogenesis.

Hamada-Kawaguchi N, Nishida Y, Yamamoto D - PLoS ONE (2015)

Effect of the Btk29AficP mutation on the localization of Btk29A, Arm and phosphotyrosine.The panels show the stage 2 (A, B, E and F) and the stage 1 (C, D, G and H) egg chambers from wild-type (A—D) and Btk29AficP mutant (E—H) ovaries doubly stained with phalloidin for actin (red in A, C, E and G) and the anti-Btk29A antibody (green in A and E) or the anti-phosphotyrosine antibody (green in C and G). The images of antibody staining are also shown in black and white in (B), (D), (F) and (H) to aid in comparisons of the localization and abundance of Btk29A or phosphotyrosine between the wild-type and Btk29AficP mutant egg chambers. Btk29A is highly enriched around ring canals and cell-cell contact regions in the wild-type chambers, whereas it is barely detectable in the Btk29AficP mutant chambers. (I—N) Arm (green) is localized along the cell-cell contact regions and ring canals in the wild-type (I) and Btk29AficP mutant (K) germaria. Actin is visualized by phalloidin staining (red). The entire stage 1 (J and L) and close-up views of ring canals in stage 1 (M and N) of the wild-type (J and M) and Btk29AficP mutant (L and N) germaria are shown. Some of the ring canals are marked with arrowheads in (A—L). Note the marked accumulations of Arm in the regions surrounding the ring canals in Btk29AficP mutants. Scale bars: 10 μm.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4372500&req=5

pone.0121484.g003: Effect of the Btk29AficP mutation on the localization of Btk29A, Arm and phosphotyrosine.The panels show the stage 2 (A, B, E and F) and the stage 1 (C, D, G and H) egg chambers from wild-type (A—D) and Btk29AficP mutant (E—H) ovaries doubly stained with phalloidin for actin (red in A, C, E and G) and the anti-Btk29A antibody (green in A and E) or the anti-phosphotyrosine antibody (green in C and G). The images of antibody staining are also shown in black and white in (B), (D), (F) and (H) to aid in comparisons of the localization and abundance of Btk29A or phosphotyrosine between the wild-type and Btk29AficP mutant egg chambers. Btk29A is highly enriched around ring canals and cell-cell contact regions in the wild-type chambers, whereas it is barely detectable in the Btk29AficP mutant chambers. (I—N) Arm (green) is localized along the cell-cell contact regions and ring canals in the wild-type (I) and Btk29AficP mutant (K) germaria. Actin is visualized by phalloidin staining (red). The entire stage 1 (J and L) and close-up views of ring canals in stage 1 (M and N) of the wild-type (J and M) and Btk29AficP mutant (L and N) germaria are shown. Some of the ring canals are marked with arrowheads in (A—L). Note the marked accumulations of Arm in the regions surrounding the ring canals in Btk29AficP mutants. Scale bars: 10 μm.
Mentions: It has been reported that germline clones for strong mutant alleles in the Btk29A locus display several distinct phenotypes, such as ring canal undergrowth [15, 17], fusome distortion [14], defects in karyosome formation [14], packaging defects [14], aberrant border cell migration (N. Hamada-Kawaguchi, unpublished data) and oocyte mislocalization [15]. In keeping with these observations, Btk29AficP mutant ovaries, expressing only the short type 1 splice form of Btk29A, were grossly aberrant as a result of the irregular shapes and sizes of germ cells and follicle cells (Figs. 1B, 1D and 1F). Moreover, the Btk29AficP egg chambers were equipped with ring canals that were much smaller than those of the wild type (Figs. 2A, 2B, 2E and 2F): mature wild-type egg chambers have ring canals of 8–10 μm in diameter (Fig. 2I), whereas those of Btk29AficP were about 3–5 μm in diameter (Fig. 2I), as reported for other Btk29A alleles that affect both type 1 and type 2 products ([15, 17] and N. Hamada-Kawaguchi, unpublished data). It is notable that the lumina of mutant ring canals appeared to be very narrow in Btk29AficP mutants, compared to the wild type (Figs. 3M and 3N).

Bottom Line: In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29AficP mutation.This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm) are located.We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology and Neurosciences, Tohoku University, Graduate School of Life Sciences, Sendai, Japan.

ABSTRACT
Drosophila Btk29A is the ortholog of mammalian Btk, a Tec family nonreceptor tyrosine kinase whose deficit causes X-linked agammaglobulinemia in humans. The Btk29AficP mutation induces multiple abnormalities in oogenesis, including the growth arrest of ring canals, large intercellular bridges that allow the flow of cytoplasm carrying maternal products essential for embryonic development from the nurse cells to the oocyte during oogenesis. In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29AficP mutation. This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm) are located. Our previous in vitro and in vivo analyses revealed that Btk29A directly phosphorylates Arm, leading to its release from DE-cadherin. In the present experiments, immunohistological analysis revealed that phosphorylation at tyrosine 150 (Y150) and Y667 of Arm was diminished in Btk29AficP mutant ring canals. Overexpression of an Arm mutant with unphosphorylatable Y150 inhibited ring canal growth. Thus Btk29A-induced Y150 phosphorylation is necessary for the normal growth of ring canals. We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.

No MeSH data available.


Related in: MedlinePlus