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Regulation of podocalyxin trafficking by Rab small GTPases in 2D and 3D epithelial cell cultures

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ABSTRACT

Epithelial polarity establishment involves transcytosis of podocalyxin to the apical domain, but its route and regulation are unclear. Here, Mrozowska and Fukuda demonstrate that different Rabs and Rab effectors coordinate podocalyxin transport during polarization in 2D and 3D structures.

No MeSH data available.


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Colocalization of endogenous PCX with EGFP-tagged Rab17 and Rab36 in 2D MDCK II cells. MDCK II cells were transfected with plasmids carrying EGFP-tagged Rab17 (A) or Rab36 (B), plated on glass-bottom dishes, and fixed with PFA at the times indicated (see also Fig. S3). The cells were then stained with anti-PCX antibody (red) and DAPI (blue). The confocal xy section (top) and the xz section (bottom), which corresponds to the dashed line in the xy section, are shown. The fourth columns show magnifications of the boxed regions in the third columns. Bars: 10 µm; (insets) 1 µm. Note that EGFP-Rab17 and PCX were well colocalized at recycling endosomes just near the nucleus 1–3 h after plating and that their colocalization was also observed just beneath the plasma membrane (indicated by arrows) 24 h after plating. In contrast, no colocalization between EGFP-Rab36 and PCX was observed during PCX trafficking. (C) Schematic summary of the Rabs that colocalize with PCX (red) at specific membrane compartments (green doted circles) in 2D MDCK II cells (see also Table 1 and Fig. S3 for details). AP, apical plasma membrane; EE, early endosome; PM, plasma membrane; TV, transport vesicle.
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fig3: Colocalization of endogenous PCX with EGFP-tagged Rab17 and Rab36 in 2D MDCK II cells. MDCK II cells were transfected with plasmids carrying EGFP-tagged Rab17 (A) or Rab36 (B), plated on glass-bottom dishes, and fixed with PFA at the times indicated (see also Fig. S3). The cells were then stained with anti-PCX antibody (red) and DAPI (blue). The confocal xy section (top) and the xz section (bottom), which corresponds to the dashed line in the xy section, are shown. The fourth columns show magnifications of the boxed regions in the third columns. Bars: 10 µm; (insets) 1 µm. Note that EGFP-Rab17 and PCX were well colocalized at recycling endosomes just near the nucleus 1–3 h after plating and that their colocalization was also observed just beneath the plasma membrane (indicated by arrows) 24 h after plating. In contrast, no colocalization between EGFP-Rab36 and PCX was observed during PCX trafficking. (C) Schematic summary of the Rabs that colocalize with PCX (red) at specific membrane compartments (green doted circles) in 2D MDCK II cells (see also Table 1 and Fig. S3 for details). AP, apical plasma membrane; EE, early endosome; PM, plasma membrane; TV, transport vesicle.

Mentions: Intracellular trafficking pathways are generally thought to be regulated by Rab small GTPases. So far, only four Rabs (Rab3B, Rab8, Rab11A, and Rab27A) are known to be engaged in PCX trafficking (Bryant et al., 2010; Gálvez-Santisteban et al., 2012), all of which mediate the docking of PCX vesicles to the apical membrane. However, nothing is known about the function of Rabs in other steps of PCX trafficking. To identify other Rab isoforms potentially involved in PCX trafficking, we performed a comprehensive colocalization screening between endogenous PCX and GFP (or Myc)-tagged Rab proteins. Because in 2D MDCK II cells PCX is transcytosed in the same manner as in 3D cells (Figs. 1 and 2), we used 2D cells in our initial colocalization screening, as they were easier to handle than Matrigel culture. We analyzed the colocalization of PCX with 60 different mammalian Rab GTPases at five consecutive time points (1, 3, 16, 24, and 48 h) representing different stages of PCX trafficking. The results of the screening are summarized in Table 1, Fig. 3, and Fig. S3.


Regulation of podocalyxin trafficking by Rab small GTPases in 2D and 3D epithelial cell cultures
Colocalization of endogenous PCX with EGFP-tagged Rab17 and Rab36 in 2D MDCK II cells. MDCK II cells were transfected with plasmids carrying EGFP-tagged Rab17 (A) or Rab36 (B), plated on glass-bottom dishes, and fixed with PFA at the times indicated (see also Fig. S3). The cells were then stained with anti-PCX antibody (red) and DAPI (blue). The confocal xy section (top) and the xz section (bottom), which corresponds to the dashed line in the xy section, are shown. The fourth columns show magnifications of the boxed regions in the third columns. Bars: 10 µm; (insets) 1 µm. Note that EGFP-Rab17 and PCX were well colocalized at recycling endosomes just near the nucleus 1–3 h after plating and that their colocalization was also observed just beneath the plasma membrane (indicated by arrows) 24 h after plating. In contrast, no colocalization between EGFP-Rab36 and PCX was observed during PCX trafficking. (C) Schematic summary of the Rabs that colocalize with PCX (red) at specific membrane compartments (green doted circles) in 2D MDCK II cells (see also Table 1 and Fig. S3 for details). AP, apical plasma membrane; EE, early endosome; PM, plasma membrane; TV, transport vesicle.
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fig3: Colocalization of endogenous PCX with EGFP-tagged Rab17 and Rab36 in 2D MDCK II cells. MDCK II cells were transfected with plasmids carrying EGFP-tagged Rab17 (A) or Rab36 (B), plated on glass-bottom dishes, and fixed with PFA at the times indicated (see also Fig. S3). The cells were then stained with anti-PCX antibody (red) and DAPI (blue). The confocal xy section (top) and the xz section (bottom), which corresponds to the dashed line in the xy section, are shown. The fourth columns show magnifications of the boxed regions in the third columns. Bars: 10 µm; (insets) 1 µm. Note that EGFP-Rab17 and PCX were well colocalized at recycling endosomes just near the nucleus 1–3 h after plating and that their colocalization was also observed just beneath the plasma membrane (indicated by arrows) 24 h after plating. In contrast, no colocalization between EGFP-Rab36 and PCX was observed during PCX trafficking. (C) Schematic summary of the Rabs that colocalize with PCX (red) at specific membrane compartments (green doted circles) in 2D MDCK II cells (see also Table 1 and Fig. S3 for details). AP, apical plasma membrane; EE, early endosome; PM, plasma membrane; TV, transport vesicle.
Mentions: Intracellular trafficking pathways are generally thought to be regulated by Rab small GTPases. So far, only four Rabs (Rab3B, Rab8, Rab11A, and Rab27A) are known to be engaged in PCX trafficking (Bryant et al., 2010; Gálvez-Santisteban et al., 2012), all of which mediate the docking of PCX vesicles to the apical membrane. However, nothing is known about the function of Rabs in other steps of PCX trafficking. To identify other Rab isoforms potentially involved in PCX trafficking, we performed a comprehensive colocalization screening between endogenous PCX and GFP (or Myc)-tagged Rab proteins. Because in 2D MDCK II cells PCX is transcytosed in the same manner as in 3D cells (Figs. 1 and 2), we used 2D cells in our initial colocalization screening, as they were easier to handle than Matrigel culture. We analyzed the colocalization of PCX with 60 different mammalian Rab GTPases at five consecutive time points (1, 3, 16, 24, and 48 h) representing different stages of PCX trafficking. The results of the screening are summarized in Table 1, Fig. 3, and Fig. S3.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Epithelial polarity establishment involves transcytosis of podocalyxin to the apical domain, but its route and regulation are unclear. Here, Mrozowska and Fukuda demonstrate that different Rabs and Rab effectors coordinate podocalyxin transport during polarization in 2D and 3D structures.

No MeSH data available.


Related in: MedlinePlus