Limits...
Membrane protein location-dependent regulation by PI3K (III) and rabenosyn-5 in Drosophila wing cells.

Abe M, Setoguchi Y, Tanaka T, Awano W, Takahashi K, Ueda R, Nakamura A, Goto S - PLoS ONE (2009)

Bottom Line: While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development.In this study, we examined in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) in Drosophila wing primordial cells, which are polarized along the apical-basal axis.These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes.

View Article: PubMed Central - PubMed

Affiliation: Glycobiology and Glycotechnology Research Group, Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan.

ABSTRACT
The class III phosphatidylinositol-3 kinase (PI3K (III)) regulates intracellular vesicular transport at multiple steps through the production of phosphatidylinositol-3-phosphate (PI(3)P). While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development. In this study, we examined in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) in Drosophila wing primordial cells, which are polarized along the apical-basal axis. Knockdown of the PI3K (III) subunit Vps15 resulted in an accumulation of the apical junctional proteins DE-cadherin and Flamingo and also the basal membrane protein beta-integrin in intracellular vesicles. By contrast, knockdown of PI3K (III) increased lateral membrane-localized Fasciclin III (Fas III). Importantly, loss-of-function mutation of Rbsn-5 recapitulated the aberrant localization phenotypes of beta-integrin and Fas III, but not those of DE-cadherin and Flamingo. These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes.

Show MeSH

Related in: MedlinePlus

Gal4/UAS mediated RNA interference in Drosophila.(A) Schematic representation of Drosophila phosphatidylinosytol 3 kinase class III (PI3K(III)) composed of dVps34 (a catalytic subunit, CG5373) and dVps15 (an adaptor subunit, CG9746). PI3K C2 domain, PI kinase conserved domain (PIK domain), PI3K catalytic domain, serine threonine protein kinase domain, WD 40 domain and N-terminal myristoylation are indicated. Underlining indicates regions used to construct dsRNA inducible vectors. (B) dVps34 and dVps15 mRNA abundance were reduced by transfection of the respective dsRNA (+) in S2 cells. RNAs was quantified by RT-PCR. γ-tubulin was used as a control. (C) Adult wings in which dsRNAs for either dVps34 (dVps34 RNAi) or dVps15 (dVps15 RNAi) were expressed by the sd-Gal4 driver. The severe malformation was completely rescued by co-expression of wild-type cDNA with dsRNA for dVps34 (dVps34 Rescue), compared with the wild-type wing (Control). (D) Confocal immunodetection showing XY and XZ sections in the plane of pupal wings at 32 h APF. Double-headed arrows indicate the regions where only GFP-2xFYVE, or both GFP-2xFYVE and dsRNA for dVps15, were expressed using the dpp-Gal4 driver. GFP-2xFYVE (green) was localized to dot-like structures in the wild type but dispersed in the cytoplasm in the dVps15-knockdown cells. Wing hairs stained by rhodamine phalloidin (red) were pointed and orderly in the wild type but deformed and irregularly pointed in the dVps15-knockdown cells. The scale bars represent 10 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2749332&req=5

pone-0007306-g001: Gal4/UAS mediated RNA interference in Drosophila.(A) Schematic representation of Drosophila phosphatidylinosytol 3 kinase class III (PI3K(III)) composed of dVps34 (a catalytic subunit, CG5373) and dVps15 (an adaptor subunit, CG9746). PI3K C2 domain, PI kinase conserved domain (PIK domain), PI3K catalytic domain, serine threonine protein kinase domain, WD 40 domain and N-terminal myristoylation are indicated. Underlining indicates regions used to construct dsRNA inducible vectors. (B) dVps34 and dVps15 mRNA abundance were reduced by transfection of the respective dsRNA (+) in S2 cells. RNAs was quantified by RT-PCR. γ-tubulin was used as a control. (C) Adult wings in which dsRNAs for either dVps34 (dVps34 RNAi) or dVps15 (dVps15 RNAi) were expressed by the sd-Gal4 driver. The severe malformation was completely rescued by co-expression of wild-type cDNA with dsRNA for dVps34 (dVps34 Rescue), compared with the wild-type wing (Control). (D) Confocal immunodetection showing XY and XZ sections in the plane of pupal wings at 32 h APF. Double-headed arrows indicate the regions where only GFP-2xFYVE, or both GFP-2xFYVE and dsRNA for dVps15, were expressed using the dpp-Gal4 driver. GFP-2xFYVE (green) was localized to dot-like structures in the wild type but dispersed in the cytoplasm in the dVps15-knockdown cells. Wing hairs stained by rhodamine phalloidin (red) were pointed and orderly in the wild type but deformed and irregularly pointed in the dVps15-knockdown cells. The scale bars represent 10 µm.

Mentions: Drosophila PI3K (III) consists of two essential components, the catalytic subunit dVps34/PI3K59F (CG5373) and the anchoring subunit dVps15/p150 (CG9746 (see Fig. 1A). To analyze the function of PI3K (III), transgenic fly strains were generated harboring ∼500 bp of inverted cDNA fragment repeats, corresponding to the PI3K (III) subunits, under the control of the GAL4-responsive UAS. Crossing these strains with appropriate Gal4 driver strains induces a hairpin type double strand RNA (dsRNA) in a Gal4-dependent manner, both temporally and spatially. Accordingly, dsRNAs in whole or restricted regions of the wing primordia were induced using scalloped-Gal4 (sd-Gal4) or decapentaplegic-Gal4 (dpp-Gal4) driver strains, respectively.


Membrane protein location-dependent regulation by PI3K (III) and rabenosyn-5 in Drosophila wing cells.

Abe M, Setoguchi Y, Tanaka T, Awano W, Takahashi K, Ueda R, Nakamura A, Goto S - PLoS ONE (2009)

Gal4/UAS mediated RNA interference in Drosophila.(A) Schematic representation of Drosophila phosphatidylinosytol 3 kinase class III (PI3K(III)) composed of dVps34 (a catalytic subunit, CG5373) and dVps15 (an adaptor subunit, CG9746). PI3K C2 domain, PI kinase conserved domain (PIK domain), PI3K catalytic domain, serine threonine protein kinase domain, WD 40 domain and N-terminal myristoylation are indicated. Underlining indicates regions used to construct dsRNA inducible vectors. (B) dVps34 and dVps15 mRNA abundance were reduced by transfection of the respective dsRNA (+) in S2 cells. RNAs was quantified by RT-PCR. γ-tubulin was used as a control. (C) Adult wings in which dsRNAs for either dVps34 (dVps34 RNAi) or dVps15 (dVps15 RNAi) were expressed by the sd-Gal4 driver. The severe malformation was completely rescued by co-expression of wild-type cDNA with dsRNA for dVps34 (dVps34 Rescue), compared with the wild-type wing (Control). (D) Confocal immunodetection showing XY and XZ sections in the plane of pupal wings at 32 h APF. Double-headed arrows indicate the regions where only GFP-2xFYVE, or both GFP-2xFYVE and dsRNA for dVps15, were expressed using the dpp-Gal4 driver. GFP-2xFYVE (green) was localized to dot-like structures in the wild type but dispersed in the cytoplasm in the dVps15-knockdown cells. Wing hairs stained by rhodamine phalloidin (red) were pointed and orderly in the wild type but deformed and irregularly pointed in the dVps15-knockdown cells. The scale bars represent 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2749332&req=5

pone-0007306-g001: Gal4/UAS mediated RNA interference in Drosophila.(A) Schematic representation of Drosophila phosphatidylinosytol 3 kinase class III (PI3K(III)) composed of dVps34 (a catalytic subunit, CG5373) and dVps15 (an adaptor subunit, CG9746). PI3K C2 domain, PI kinase conserved domain (PIK domain), PI3K catalytic domain, serine threonine protein kinase domain, WD 40 domain and N-terminal myristoylation are indicated. Underlining indicates regions used to construct dsRNA inducible vectors. (B) dVps34 and dVps15 mRNA abundance were reduced by transfection of the respective dsRNA (+) in S2 cells. RNAs was quantified by RT-PCR. γ-tubulin was used as a control. (C) Adult wings in which dsRNAs for either dVps34 (dVps34 RNAi) or dVps15 (dVps15 RNAi) were expressed by the sd-Gal4 driver. The severe malformation was completely rescued by co-expression of wild-type cDNA with dsRNA for dVps34 (dVps34 Rescue), compared with the wild-type wing (Control). (D) Confocal immunodetection showing XY and XZ sections in the plane of pupal wings at 32 h APF. Double-headed arrows indicate the regions where only GFP-2xFYVE, or both GFP-2xFYVE and dsRNA for dVps15, were expressed using the dpp-Gal4 driver. GFP-2xFYVE (green) was localized to dot-like structures in the wild type but dispersed in the cytoplasm in the dVps15-knockdown cells. Wing hairs stained by rhodamine phalloidin (red) were pointed and orderly in the wild type but deformed and irregularly pointed in the dVps15-knockdown cells. The scale bars represent 10 µm.
Mentions: Drosophila PI3K (III) consists of two essential components, the catalytic subunit dVps34/PI3K59F (CG5373) and the anchoring subunit dVps15/p150 (CG9746 (see Fig. 1A). To analyze the function of PI3K (III), transgenic fly strains were generated harboring ∼500 bp of inverted cDNA fragment repeats, corresponding to the PI3K (III) subunits, under the control of the GAL4-responsive UAS. Crossing these strains with appropriate Gal4 driver strains induces a hairpin type double strand RNA (dsRNA) in a Gal4-dependent manner, both temporally and spatially. Accordingly, dsRNAs in whole or restricted regions of the wing primordia were induced using scalloped-Gal4 (sd-Gal4) or decapentaplegic-Gal4 (dpp-Gal4) driver strains, respectively.

Bottom Line: While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development.In this study, we examined in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) in Drosophila wing primordial cells, which are polarized along the apical-basal axis.These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes.

View Article: PubMed Central - PubMed

Affiliation: Glycobiology and Glycotechnology Research Group, Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan.

ABSTRACT
The class III phosphatidylinositol-3 kinase (PI3K (III)) regulates intracellular vesicular transport at multiple steps through the production of phosphatidylinositol-3-phosphate (PI(3)P). While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development. In this study, we examined in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) in Drosophila wing primordial cells, which are polarized along the apical-basal axis. Knockdown of the PI3K (III) subunit Vps15 resulted in an accumulation of the apical junctional proteins DE-cadherin and Flamingo and also the basal membrane protein beta-integrin in intracellular vesicles. By contrast, knockdown of PI3K (III) increased lateral membrane-localized Fasciclin III (Fas III). Importantly, loss-of-function mutation of Rbsn-5 recapitulated the aberrant localization phenotypes of beta-integrin and Fas III, but not those of DE-cadherin and Flamingo. These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes.

Show MeSH
Related in: MedlinePlus