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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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Endocytotic trafficking is compromised by knockdown of PI3K (III).(A) GFP-2xFYVE co-localized with Rab7 and Lysotracker but not with GM130, Rab5 or Rab11. Arrowheads point to the GFP-2xFYVE-positive compartments with Rab7 and Lysotracker. (B) The numbers of Rab5-, Rab7- and Nuf-positive endosomes (red) were increased in the dVps15-knockdown cells (left sides of white lines, indicated by double-headed arrows) compared with the wild type cells (right sides of white lines). The GFP-2xFYVE (green) and dsRNA for dVps15 were simultaneously expressed by dpp-Gal4 driver. (C) Upper panels: Trafficking of TR-D from the plasma membrane to the lysosome was monitored by double labeling of TR-D and GFP-LAMP1 (lysosome marker, green) in the wing discs. Wing discs of the wild type or dVps15 knockdown were pulse labeled with TR-D for 5 min and chased in Schneider's medium for the indicated times. The wing discs were then fixed and analyzed by confocal microscopy. Arrows show the co-localization of TR-D and GFP- LAMP1. The scale bars represent 10 µm. Lower graph: The ratios of co-localized TR-D to GFP-LAMP1 in the wild or dVps15 knockdown discs (n = 89∼152).
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pone-0007306-g002: Endocytotic trafficking is compromised by knockdown of PI3K (III).(A) GFP-2xFYVE co-localized with Rab7 and Lysotracker but not with GM130, Rab5 or Rab11. Arrowheads point to the GFP-2xFYVE-positive compartments with Rab7 and Lysotracker. (B) The numbers of Rab5-, Rab7- and Nuf-positive endosomes (red) were increased in the dVps15-knockdown cells (left sides of white lines, indicated by double-headed arrows) compared with the wild type cells (right sides of white lines). The GFP-2xFYVE (green) and dsRNA for dVps15 were simultaneously expressed by dpp-Gal4 driver. (C) Upper panels: Trafficking of TR-D from the plasma membrane to the lysosome was monitored by double labeling of TR-D and GFP-LAMP1 (lysosome marker, green) in the wing discs. Wing discs of the wild type or dVps15 knockdown were pulse labeled with TR-D for 5 min and chased in Schneider's medium for the indicated times. The wing discs were then fixed and analyzed by confocal microscopy. Arrows show the co-localization of TR-D and GFP- LAMP1. The scale bars represent 10 µm. Lower graph: The ratios of co-localized TR-D to GFP-LAMP1 in the wild or dVps15 knockdown discs (n = 89∼152).

Mentions: The Rab family of GTPases plays pivotal roles in the regulation of vesicular trafficking, the Rab5, Rab7 and Rab11 members contributing to the formation and dynamics of early endosomes, late endosomes and recycling endosomes, respectively [41]. It seems likely that PI3K (III) products might co-localize with these Rab family proteins since they are known to physically associate with Rab5 [10], and possibly with Rab7 [42]. Indeed, GFP-2xFYVE has been shown to localize with Rab5-positive endosomes, when expressed in Drosophila S2 and neural cells [40]. We therefore examined which endosomal compartments GFP-2xFYVE is localized to when expressed in larval wing cells. In larval wing disc cells GFP-2xFYVE was localized mostly to Rab7-positive endosomes, but not to Rab5- or Rab11-positive endosomes (Fig. 2A). In addition, some of the GFP-2xFYVE-positive endosomes were stained with Lysotracker (Fig. 2A), suggesting that they were acidic, including the late endosomes. PI3K (III) products therefore appear to localize to Rab7-positive late endosomes in these 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)

Endocytotic trafficking is compromised by knockdown of PI3K (III).(A) GFP-2xFYVE co-localized with Rab7 and Lysotracker but not with GM130, Rab5 or Rab11. Arrowheads point to the GFP-2xFYVE-positive compartments with Rab7 and Lysotracker. (B) The numbers of Rab5-, Rab7- and Nuf-positive endosomes (red) were increased in the dVps15-knockdown cells (left sides of white lines, indicated by double-headed arrows) compared with the wild type cells (right sides of white lines). The GFP-2xFYVE (green) and dsRNA for dVps15 were simultaneously expressed by dpp-Gal4 driver. (C) Upper panels: Trafficking of TR-D from the plasma membrane to the lysosome was monitored by double labeling of TR-D and GFP-LAMP1 (lysosome marker, green) in the wing discs. Wing discs of the wild type or dVps15 knockdown were pulse labeled with TR-D for 5 min and chased in Schneider's medium for the indicated times. The wing discs were then fixed and analyzed by confocal microscopy. Arrows show the co-localization of TR-D and GFP- LAMP1. The scale bars represent 10 µm. Lower graph: The ratios of co-localized TR-D to GFP-LAMP1 in the wild or dVps15 knockdown discs (n = 89∼152).
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Related In: Results  -  Collection

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pone-0007306-g002: Endocytotic trafficking is compromised by knockdown of PI3K (III).(A) GFP-2xFYVE co-localized with Rab7 and Lysotracker but not with GM130, Rab5 or Rab11. Arrowheads point to the GFP-2xFYVE-positive compartments with Rab7 and Lysotracker. (B) The numbers of Rab5-, Rab7- and Nuf-positive endosomes (red) were increased in the dVps15-knockdown cells (left sides of white lines, indicated by double-headed arrows) compared with the wild type cells (right sides of white lines). The GFP-2xFYVE (green) and dsRNA for dVps15 were simultaneously expressed by dpp-Gal4 driver. (C) Upper panels: Trafficking of TR-D from the plasma membrane to the lysosome was monitored by double labeling of TR-D and GFP-LAMP1 (lysosome marker, green) in the wing discs. Wing discs of the wild type or dVps15 knockdown were pulse labeled with TR-D for 5 min and chased in Schneider's medium for the indicated times. The wing discs were then fixed and analyzed by confocal microscopy. Arrows show the co-localization of TR-D and GFP- LAMP1. The scale bars represent 10 µm. Lower graph: The ratios of co-localized TR-D to GFP-LAMP1 in the wild or dVps15 knockdown discs (n = 89∼152).
Mentions: The Rab family of GTPases plays pivotal roles in the regulation of vesicular trafficking, the Rab5, Rab7 and Rab11 members contributing to the formation and dynamics of early endosomes, late endosomes and recycling endosomes, respectively [41]. It seems likely that PI3K (III) products might co-localize with these Rab family proteins since they are known to physically associate with Rab5 [10], and possibly with Rab7 [42]. Indeed, GFP-2xFYVE has been shown to localize with Rab5-positive endosomes, when expressed in Drosophila S2 and neural cells [40]. We therefore examined which endosomal compartments GFP-2xFYVE is localized to when expressed in larval wing cells. In larval wing disc cells GFP-2xFYVE was localized mostly to Rab7-positive endosomes, but not to Rab5- or Rab11-positive endosomes (Fig. 2A). In addition, some of the GFP-2xFYVE-positive endosomes were stained with Lysotracker (Fig. 2A), suggesting that they were acidic, including the late endosomes. PI3K (III) products therefore appear to localize to Rab7-positive late endosomes in these 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