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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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PCX colocalization with organelle markers in 2D monolayers and in 3D cysts. MDCK II cells were plated on glass bottom dishes (2D) or Matrigel-coated glass slides (3D) and fixed with PFA at the times indicated (see also Fig. S2). (A and B) Cells were costained with anti-PCX antibody (red), DAPI (blue) and an antibody against EEA1 (an early endosome marker; A), GM130 (a Golgi marker; B), or Rab11 (an RE marker; green; C). The confocal xy section (top) and the xz section (bottom), which corresponds to the dashed line in the xy section, of 2D monolayers are shown. The fourth columns show magnifications of the boxed regions in the third columns. The arrows in A show the colocalization points between EEA1 and PCX. Colocalization of PCX with each organelle marker is illustrated schematically on the right side of each panel (red, PCX; green, marker; yellow, colocalization site; and blue, nucleus). Bars: 10 µm; (insets) 1 µm.
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fig2: PCX colocalization with organelle markers in 2D monolayers and in 3D cysts. MDCK II cells were plated on glass bottom dishes (2D) or Matrigel-coated glass slides (3D) and fixed with PFA at the times indicated (see also Fig. S2). (A and B) Cells were costained with anti-PCX antibody (red), DAPI (blue) and an antibody against EEA1 (an early endosome marker; A), GM130 (a Golgi marker; B), or Rab11 (an RE marker; green; C). The confocal xy section (top) and the xz section (bottom), which corresponds to the dashed line in the xy section, of 2D monolayers are shown. The fourth columns show magnifications of the boxed regions in the third columns. The arrows in A show the colocalization points between EEA1 and PCX. Colocalization of PCX with each organelle marker is illustrated schematically on the right side of each panel (red, PCX; green, marker; yellow, colocalization site; and blue, nucleus). Bars: 10 µm; (insets) 1 µm.

Mentions: Although PCX showed similar trafficking pattern in cells polarizing under both 2D and 3D culture conditions, it is not clear whether PCX is localized to the same intermediate compartments under both conditions. To determine that, we fixed MDCK II cells growing on a glass-bottom dish and in Matrigel at consecutive time points and costained the cells with antibodies against PCX and various organelle markers. PCX showed brief colocalization with an early endosome marker EEA1 both in 2D and 3D cell cultures at the early stages of cell growth (Figs. 2 A and S2 A). PCX did not colocalize with the Golgi marker GM130 in 2D or 3D cell culture (Figs. 2 B and S2 B), suggesting that observed intracellular PCX is not freshly biosynthesized but endocytosed. Intracellular signals of PCX were still observed even in the presence of cycloheximide, an inhibitor of protein synthesis (unpublished data). Because we expected transcytosed PCX to be localized to the recycling endosomes (REs), we also analyzed the colocalization with two RE markers: Rab11 and transferrin receptor (TfR). According to our previous data, Rab11 and TfR occupy different subpopulations of recycling endosomes, with TfR localized more to the outer fraction of REs and Rab11 to the inner fraction (Kobayashi and Fukuda, 2013b). In both 2D and 3D cell cultures, PCX colocalized extensively with Rab11 but showed only limited colocalization with TfR (Figs. 2 C and S2 C). Moreover, in terminally polarized cells under both conditions, Rab11 was similarly localized just below the apical membrane (Fig. 2 C), forming the so-called apical recycling endosome (Casanova et al., 1999), whereas TfR was scattered in the cytoplasm (Fig. S2 C). This interesting observation additionally revealed that in the process of polarity establishment in MDCK II cells, only the Rab11-positive inner fraction of REs transformed into the apical RE, whereas the TfR-positive outer fraction dispersed in the cytoplasm.


Regulation of podocalyxin trafficking by Rab small GTPases in 2D and 3D epithelial cell cultures
PCX colocalization with organelle markers in 2D monolayers and in 3D cysts. MDCK II cells were plated on glass bottom dishes (2D) or Matrigel-coated glass slides (3D) and fixed with PFA at the times indicated (see also Fig. S2). (A and B) Cells were costained with anti-PCX antibody (red), DAPI (blue) and an antibody against EEA1 (an early endosome marker; A), GM130 (a Golgi marker; B), or Rab11 (an RE marker; green; C). The confocal xy section (top) and the xz section (bottom), which corresponds to the dashed line in the xy section, of 2D monolayers are shown. The fourth columns show magnifications of the boxed regions in the third columns. The arrows in A show the colocalization points between EEA1 and PCX. Colocalization of PCX with each organelle marker is illustrated schematically on the right side of each panel (red, PCX; green, marker; yellow, colocalization site; and blue, nucleus). Bars: 10 µm; (insets) 1 µm.
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fig2: PCX colocalization with organelle markers in 2D monolayers and in 3D cysts. MDCK II cells were plated on glass bottom dishes (2D) or Matrigel-coated glass slides (3D) and fixed with PFA at the times indicated (see also Fig. S2). (A and B) Cells were costained with anti-PCX antibody (red), DAPI (blue) and an antibody against EEA1 (an early endosome marker; A), GM130 (a Golgi marker; B), or Rab11 (an RE marker; green; C). The confocal xy section (top) and the xz section (bottom), which corresponds to the dashed line in the xy section, of 2D monolayers are shown. The fourth columns show magnifications of the boxed regions in the third columns. The arrows in A show the colocalization points between EEA1 and PCX. Colocalization of PCX with each organelle marker is illustrated schematically on the right side of each panel (red, PCX; green, marker; yellow, colocalization site; and blue, nucleus). Bars: 10 µm; (insets) 1 µm.
Mentions: Although PCX showed similar trafficking pattern in cells polarizing under both 2D and 3D culture conditions, it is not clear whether PCX is localized to the same intermediate compartments under both conditions. To determine that, we fixed MDCK II cells growing on a glass-bottom dish and in Matrigel at consecutive time points and costained the cells with antibodies against PCX and various organelle markers. PCX showed brief colocalization with an early endosome marker EEA1 both in 2D and 3D cell cultures at the early stages of cell growth (Figs. 2 A and S2 A). PCX did not colocalize with the Golgi marker GM130 in 2D or 3D cell culture (Figs. 2 B and S2 B), suggesting that observed intracellular PCX is not freshly biosynthesized but endocytosed. Intracellular signals of PCX were still observed even in the presence of cycloheximide, an inhibitor of protein synthesis (unpublished data). Because we expected transcytosed PCX to be localized to the recycling endosomes (REs), we also analyzed the colocalization with two RE markers: Rab11 and transferrin receptor (TfR). According to our previous data, Rab11 and TfR occupy different subpopulations of recycling endosomes, with TfR localized more to the outer fraction of REs and Rab11 to the inner fraction (Kobayashi and Fukuda, 2013b). In both 2D and 3D cell cultures, PCX colocalized extensively with Rab11 but showed only limited colocalization with TfR (Figs. 2 C and S2 C). Moreover, in terminally polarized cells under both conditions, Rab11 was similarly localized just below the apical membrane (Fig. 2 C), forming the so-called apical recycling endosome (Casanova et al., 1999), whereas TfR was scattered in the cytoplasm (Fig. S2 C). This interesting observation additionally revealed that in the process of polarity establishment in MDCK II cells, only the Rab11-positive inner fraction of REs transformed into the apical RE, whereas the TfR-positive outer fraction dispersed in the cytoplasm.

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