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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.

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Rbsn-5 was selectively required for the correct localization of FasIII and β-integrin.(A) Horizontal sections of 27 h APF pupal wings, expressing GFP-2xFYVE, with or without dsRNA, for Rbsn-5 (lower or upper panels, respectively). Images were obtained for repeated 1 µm sections from the apical side. Merged horizontal sections in the plane of the ZA and septate junctions are shown as Apical and those in the plane between the BLPM and bHAJ are indicated as BLPM-bHAJ. β-integrin was mostly localized to the basal plasma membrane and formed large clusters. In Rbsn-5 knockdown cells, β-integrin was rarely localized to the basal plasma membrane and there was only a small amount of accumulation in the apical region. Fas III was localized to the SJ, whereas in Rbsn-5 knockdown cells it also accumulated in the BLPM. In addition, two types of GFP-2xFYVE-positive endosomes were observed as dot-like small structures in the apical regions and vesicle-like large structures in basal regions in the wild type. In Rbsn-5 knockdown cells, very few large endosomes were observed in the basal regions. Note that the large endosomes were very close to the BLPM membrane and frequently contained Fas III. (B) Vertical sections of the pupal wings expressing GFP-2xFYVE (green) with dsRNA for Rbsn-5. (left) β-integrin (red) was not localized to the basal junctions in the knockdown cells where the GFP-2xFYVE is expressed (green). (right) Localization of Fas III (red) extended to the BLPM in the knockdown cells (green). The normal distributions of these proteins were presented in the internal control regions where GFP-2xFYVE was not expressed. (C) Confocal immunodetection showing XY (a, c, e, f, g and h) and XZ (b and d) sections in the plane of the pupal wings at 30–32 hr APF. Cells that are encircled by white lines were GFP-negative and mutant for Rbsn-5C241. β-integrin (red in a and b) accumulated intracellularly, as compared with the wild-type cells surrounding the mutant cells. Fas III (red in c and d) accumulated in the whole basolateral plasma membrane, as compared with the GFP-positive wild-type cells. Fmi (red in e), DE-cadherin (red in f), Arm (red in g) and wing hairs (red in h) were normal. The scale bars represent 10 µm.
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pone-0007306-g006: Rbsn-5 was selectively required for the correct localization of FasIII and β-integrin.(A) Horizontal sections of 27 h APF pupal wings, expressing GFP-2xFYVE, with or without dsRNA, for Rbsn-5 (lower or upper panels, respectively). Images were obtained for repeated 1 µm sections from the apical side. Merged horizontal sections in the plane of the ZA and septate junctions are shown as Apical and those in the plane between the BLPM and bHAJ are indicated as BLPM-bHAJ. β-integrin was mostly localized to the basal plasma membrane and formed large clusters. In Rbsn-5 knockdown cells, β-integrin was rarely localized to the basal plasma membrane and there was only a small amount of accumulation in the apical region. Fas III was localized to the SJ, whereas in Rbsn-5 knockdown cells it also accumulated in the BLPM. In addition, two types of GFP-2xFYVE-positive endosomes were observed as dot-like small structures in the apical regions and vesicle-like large structures in basal regions in the wild type. In Rbsn-5 knockdown cells, very few large endosomes were observed in the basal regions. Note that the large endosomes were very close to the BLPM membrane and frequently contained Fas III. (B) Vertical sections of the pupal wings expressing GFP-2xFYVE (green) with dsRNA for Rbsn-5. (left) β-integrin (red) was not localized to the basal junctions in the knockdown cells where the GFP-2xFYVE is expressed (green). (right) Localization of Fas III (red) extended to the BLPM in the knockdown cells (green). The normal distributions of these proteins were presented in the internal control regions where GFP-2xFYVE was not expressed. (C) Confocal immunodetection showing XY (a, c, e, f, g and h) and XZ (b and d) sections in the plane of the pupal wings at 30–32 hr APF. Cells that are encircled by white lines were GFP-negative and mutant for Rbsn-5C241. β-integrin (red in a and b) accumulated intracellularly, as compared with the wild-type cells surrounding the mutant cells. Fas III (red in c and d) accumulated in the whole basolateral plasma membrane, as compared with the GFP-positive wild-type cells. Fmi (red in e), DE-cadherin (red in f), Arm (red in g) and wing hairs (red in h) were normal. The scale bars represent 10 µm.

Mentions: The above results indicate that PI3K (III) is required for the degradation and correct localization of Fmi, DE-cadherin, Fas III and β-integrin. To identify the factor under the control of PI(3)P, we selected genes from the Drosophila genome database that contain FYVE and/or PX domains, since these domains are known to bind to PI(3)P. The dsRNAs were then expressed, corresponding to each of the selected genes in the wing discs, and the effect on the localization of these membrane proteins determined. From this screen, we found that a Drosophila Rbsn-5 knockdown influenced the localization of Fas III and β-integrin. In wild type cells, β-integrin was found to localize at or near the basal membrane as large clusters but was absent from the apical region at 27 h APF (Fig. 6A). However, knockdown of Rbsn-5 markedly reduced the amount of β-integrin localized at or near the basal membrane and slightly increased the amount of intracellular vesicles located in the apical region (Fig. 6A and B). Furthermore, at 30 h APF for wild type cells, Fas III was mainly localized in the apical region of the lateral plasma membrane, while only a small fraction of Fas III was localized in the basolateral region (Fig. 6A). By contrast, when Rbsn-5 was knocked down, Fas III was also found at the basal region of the lateral plasma membrane (Fig. 6A and B). These Rbsn-5 knockdown cell phenotypes were reminiscent of those of the PI3K (III) knockdown cells.


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)

Rbsn-5 was selectively required for the correct localization of FasIII and β-integrin.(A) Horizontal sections of 27 h APF pupal wings, expressing GFP-2xFYVE, with or without dsRNA, for Rbsn-5 (lower or upper panels, respectively). Images were obtained for repeated 1 µm sections from the apical side. Merged horizontal sections in the plane of the ZA and septate junctions are shown as Apical and those in the plane between the BLPM and bHAJ are indicated as BLPM-bHAJ. β-integrin was mostly localized to the basal plasma membrane and formed large clusters. In Rbsn-5 knockdown cells, β-integrin was rarely localized to the basal plasma membrane and there was only a small amount of accumulation in the apical region. Fas III was localized to the SJ, whereas in Rbsn-5 knockdown cells it also accumulated in the BLPM. In addition, two types of GFP-2xFYVE-positive endosomes were observed as dot-like small structures in the apical regions and vesicle-like large structures in basal regions in the wild type. In Rbsn-5 knockdown cells, very few large endosomes were observed in the basal regions. Note that the large endosomes were very close to the BLPM membrane and frequently contained Fas III. (B) Vertical sections of the pupal wings expressing GFP-2xFYVE (green) with dsRNA for Rbsn-5. (left) β-integrin (red) was not localized to the basal junctions in the knockdown cells where the GFP-2xFYVE is expressed (green). (right) Localization of Fas III (red) extended to the BLPM in the knockdown cells (green). The normal distributions of these proteins were presented in the internal control regions where GFP-2xFYVE was not expressed. (C) Confocal immunodetection showing XY (a, c, e, f, g and h) and XZ (b and d) sections in the plane of the pupal wings at 30–32 hr APF. Cells that are encircled by white lines were GFP-negative and mutant for Rbsn-5C241. β-integrin (red in a and b) accumulated intracellularly, as compared with the wild-type cells surrounding the mutant cells. Fas III (red in c and d) accumulated in the whole basolateral plasma membrane, as compared with the GFP-positive wild-type cells. Fmi (red in e), DE-cadherin (red in f), Arm (red in g) and wing hairs (red in h) were normal. The scale bars represent 10 µm.
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Related In: Results  -  Collection

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pone-0007306-g006: Rbsn-5 was selectively required for the correct localization of FasIII and β-integrin.(A) Horizontal sections of 27 h APF pupal wings, expressing GFP-2xFYVE, with or without dsRNA, for Rbsn-5 (lower or upper panels, respectively). Images were obtained for repeated 1 µm sections from the apical side. Merged horizontal sections in the plane of the ZA and septate junctions are shown as Apical and those in the plane between the BLPM and bHAJ are indicated as BLPM-bHAJ. β-integrin was mostly localized to the basal plasma membrane and formed large clusters. In Rbsn-5 knockdown cells, β-integrin was rarely localized to the basal plasma membrane and there was only a small amount of accumulation in the apical region. Fas III was localized to the SJ, whereas in Rbsn-5 knockdown cells it also accumulated in the BLPM. In addition, two types of GFP-2xFYVE-positive endosomes were observed as dot-like small structures in the apical regions and vesicle-like large structures in basal regions in the wild type. In Rbsn-5 knockdown cells, very few large endosomes were observed in the basal regions. Note that the large endosomes were very close to the BLPM membrane and frequently contained Fas III. (B) Vertical sections of the pupal wings expressing GFP-2xFYVE (green) with dsRNA for Rbsn-5. (left) β-integrin (red) was not localized to the basal junctions in the knockdown cells where the GFP-2xFYVE is expressed (green). (right) Localization of Fas III (red) extended to the BLPM in the knockdown cells (green). The normal distributions of these proteins were presented in the internal control regions where GFP-2xFYVE was not expressed. (C) Confocal immunodetection showing XY (a, c, e, f, g and h) and XZ (b and d) sections in the plane of the pupal wings at 30–32 hr APF. Cells that are encircled by white lines were GFP-negative and mutant for Rbsn-5C241. β-integrin (red in a and b) accumulated intracellularly, as compared with the wild-type cells surrounding the mutant cells. Fas III (red in c and d) accumulated in the whole basolateral plasma membrane, as compared with the GFP-positive wild-type cells. Fmi (red in e), DE-cadherin (red in f), Arm (red in g) and wing hairs (red in h) were normal. The scale bars represent 10 µm.
Mentions: The above results indicate that PI3K (III) is required for the degradation and correct localization of Fmi, DE-cadherin, Fas III and β-integrin. To identify the factor under the control of PI(3)P, we selected genes from the Drosophila genome database that contain FYVE and/or PX domains, since these domains are known to bind to PI(3)P. The dsRNAs were then expressed, corresponding to each of the selected genes in the wing discs, and the effect on the localization of these membrane proteins determined. From this screen, we found that a Drosophila Rbsn-5 knockdown influenced the localization of Fas III and β-integrin. In wild type cells, β-integrin was found to localize at or near the basal membrane as large clusters but was absent from the apical region at 27 h APF (Fig. 6A). However, knockdown of Rbsn-5 markedly reduced the amount of β-integrin localized at or near the basal membrane and slightly increased the amount of intracellular vesicles located in the apical region (Fig. 6A and B). Furthermore, at 30 h APF for wild type cells, Fas III was mainly localized in the apical region of the lateral plasma membrane, while only a small fraction of Fas III was localized in the basolateral region (Fig. 6A). By contrast, when Rbsn-5 was knocked down, Fas III was also found at the basal region of the lateral plasma membrane (Fig. 6A and B). These Rbsn-5 knockdown cell phenotypes were reminiscent of those of the PI3K (III) knockdown cells.

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