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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 is a PI(3)P target protein.(A) Rbsn-5 (red) was co-localized in the pupal wing discs, mainly with GFP-2xFYVE, Rab5 and Rab7 (arrowheads), but not with Rab11. The scale bars represent 20 µm. (B) Rbsn-5 (red) was clearly reduced in the dVps15-knockdown region (the left sides of the dotted lines). In the knockdown region, DE-Cadherin (green) accumulated intracellularly as shown in Figure 4. (C) Adult wings in which dsRNAs were expressed by dpp-Gal4 driver. Malformation was observed near the third vein where the dsRNAs for dVps15, dVps34 or Rbsn-5 were expressed. This malformation was synergistically enhanced by co-expression of dsRNAs for both dVps15 and dVps34 or both dVps15 and Rbsn-5, whereas no such enhancement was observed by co-expression of dsRNAs for both PI3K class I and Rbsn-5 or both PI3K class I and dVps34. (D) Horizontal (upper) and vertical (lower) sections of the pupal wings expressing dsRNAs for Rbsn-5 and/or dVps15. β-integrin (red) was not localized in the double knockdown region (indicated by double headed arrow in the left panel) whereas marginal defects were observed in the single knockdown (indicated by double headed arrows in the middle and right panels). The right sides of the panels represent the internal control where β-integrin was normally localized to the basal junctions. The scale bars represent 10 µm.
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pone-0007306-g008: Rbsn-5 is a PI(3)P target protein.(A) Rbsn-5 (red) was co-localized in the pupal wing discs, mainly with GFP-2xFYVE, Rab5 and Rab7 (arrowheads), but not with Rab11. The scale bars represent 20 µm. (B) Rbsn-5 (red) was clearly reduced in the dVps15-knockdown region (the left sides of the dotted lines). In the knockdown region, DE-Cadherin (green) accumulated intracellularly as shown in Figure 4. (C) Adult wings in which dsRNAs were expressed by dpp-Gal4 driver. Malformation was observed near the third vein where the dsRNAs for dVps15, dVps34 or Rbsn-5 were expressed. This malformation was synergistically enhanced by co-expression of dsRNAs for both dVps15 and dVps34 or both dVps15 and Rbsn-5, whereas no such enhancement was observed by co-expression of dsRNAs for both PI3K class I and Rbsn-5 or both PI3K class I and dVps34. (D) Horizontal (upper) and vertical (lower) sections of the pupal wings expressing dsRNAs for Rbsn-5 and/or dVps15. β-integrin (red) was not localized in the double knockdown region (indicated by double headed arrow in the left panel) whereas marginal defects were observed in the single knockdown (indicated by double headed arrows in the middle and right panels). The right sides of the panels represent the internal control where β-integrin was normally localized to the basal junctions. The scale bars represent 10 µm.

Mentions: In addition to PI(3)P, Rbsn-5 has been shown to associate with Rab5 and Rab4 and to be controlled by these Rab family small GTPases, which govern endosome fusion and recycling processes, respectively [31]. Drosophila Rbsn-5 can bind to the GTP form of Rab5, but not to Rab4 or Rab7 in vitro [35], [46]. Although Rbsn-5 has been proposed to function at Rab5 or Rab4-positive endosomes in mammalian cells, we found that it was localized not only at the Rab5-positive endosomes but also the Rab7-positive endosomes, whereas it was absent from Rab11-positive endosomes (Fig. 8A). This supports the notion that Rbsn-5 is involved in the sorting to late endosomes in the degradation pathway.


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 is a PI(3)P target protein.(A) Rbsn-5 (red) was co-localized in the pupal wing discs, mainly with GFP-2xFYVE, Rab5 and Rab7 (arrowheads), but not with Rab11. The scale bars represent 20 µm. (B) Rbsn-5 (red) was clearly reduced in the dVps15-knockdown region (the left sides of the dotted lines). In the knockdown region, DE-Cadherin (green) accumulated intracellularly as shown in Figure 4. (C) Adult wings in which dsRNAs were expressed by dpp-Gal4 driver. Malformation was observed near the third vein where the dsRNAs for dVps15, dVps34 or Rbsn-5 were expressed. This malformation was synergistically enhanced by co-expression of dsRNAs for both dVps15 and dVps34 or both dVps15 and Rbsn-5, whereas no such enhancement was observed by co-expression of dsRNAs for both PI3K class I and Rbsn-5 or both PI3K class I and dVps34. (D) Horizontal (upper) and vertical (lower) sections of the pupal wings expressing dsRNAs for Rbsn-5 and/or dVps15. β-integrin (red) was not localized in the double knockdown region (indicated by double headed arrow in the left panel) whereas marginal defects were observed in the single knockdown (indicated by double headed arrows in the middle and right panels). The right sides of the panels represent the internal control where β-integrin was normally localized to the basal junctions. The scale bars represent 10 µm.
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Related In: Results  -  Collection

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pone-0007306-g008: Rbsn-5 is a PI(3)P target protein.(A) Rbsn-5 (red) was co-localized in the pupal wing discs, mainly with GFP-2xFYVE, Rab5 and Rab7 (arrowheads), but not with Rab11. The scale bars represent 20 µm. (B) Rbsn-5 (red) was clearly reduced in the dVps15-knockdown region (the left sides of the dotted lines). In the knockdown region, DE-Cadherin (green) accumulated intracellularly as shown in Figure 4. (C) Adult wings in which dsRNAs were expressed by dpp-Gal4 driver. Malformation was observed near the third vein where the dsRNAs for dVps15, dVps34 or Rbsn-5 were expressed. This malformation was synergistically enhanced by co-expression of dsRNAs for both dVps15 and dVps34 or both dVps15 and Rbsn-5, whereas no such enhancement was observed by co-expression of dsRNAs for both PI3K class I and Rbsn-5 or both PI3K class I and dVps34. (D) Horizontal (upper) and vertical (lower) sections of the pupal wings expressing dsRNAs for Rbsn-5 and/or dVps15. β-integrin (red) was not localized in the double knockdown region (indicated by double headed arrow in the left panel) whereas marginal defects were observed in the single knockdown (indicated by double headed arrows in the middle and right panels). The right sides of the panels represent the internal control where β-integrin was normally localized to the basal junctions. The scale bars represent 10 µm.
Mentions: In addition to PI(3)P, Rbsn-5 has been shown to associate with Rab5 and Rab4 and to be controlled by these Rab family small GTPases, which govern endosome fusion and recycling processes, respectively [31]. Drosophila Rbsn-5 can bind to the GTP form of Rab5, but not to Rab4 or Rab7 in vitro [35], [46]. Although Rbsn-5 has been proposed to function at Rab5 or Rab4-positive endosomes in mammalian cells, we found that it was localized not only at the Rab5-positive endosomes but also the Rab7-positive endosomes, whereas it was absent from Rab11-positive endosomes (Fig. 8A). This supports the notion that Rbsn-5 is involved in the sorting to late endosomes in the degradation pathway.

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