Figure 1. Podocalyxin is excluded from... | Rockefeller University Press

$Figure 1. Podocalyxin is excluded from the CMD before emergence of a cilium. (A) MDCK cells expressing GFP-PODXL were grown on filters for 8 d and were fixed and stained with antiacetylated tubulin to visualize primary cilia and phalloidin to visualize F-actin. Bar, 20 µm. (B) Cells expressing GFP-PODXL and RFP-NHERF1 were fixed and stained with antiacetylated tubulin 4 or 8 d after seeding on filters. GFP-PODXL and RFP-NHERF1 patterns are similar between days 4 and 8, but few cilia are present on day 4. Bar, 20 µm. (C) Live cells expressing GFP-PODXL were imaged and individually scored for the appearance of a GFP-PODXL exclusion zone in the center of the apical membrane. Three fields of cells were counted for each of two experiments, and the percentage of cells with apparent exclusion zones appears on each image. On day 1, only 16/150 cells appeared to have a GFP-PODXL exclusion zone. The fraction rose on day 2 (46/214) and dramatically increased on day 3 (166/257) before reaching a plateau on day 4 (182/250). Images were collected with identical microscope settings. Bar, 10 µm. (D) 24 h after seeding on a filter, cells were fixed and stained with anti–γ-tubulin to visualize centrioles and anti-gp135 to visualize endogenous podocalyxin. Centrioles are aligned below the center of the apical membrane before podocalyxin is excluded from the CMD. Small podocalyxin exclusion zones are only seen in fixed samples. Images represent a single confocal plane. Bar, 20 µm. (E) SEM imaging of cells 4 d after seeding surprisingly shows microvilli in the CMD. After 8 d, an area at the base of primary cilia (arrowheads) is free of microvilli. Images in A–C are projected z stacks. Bars: (left) 5 µm; (right) 2 µm.$

Figure 1.

Podocalyxin is excluded from the CMD before emergence of a cilium. (A) MDCK cells expressing GFP-PODXL were grown on filters for 8 d and were fixed and stained with antiacetylated tubulin to visualize primary cilia and phalloidin to visualize F-actin. Bar, 20 µm. (B) Cells expressing GFP-PODXL and RFP-NHERF1 were fixed and stained with antiacetylated tubulin 4 or 8 d after seeding on filters. GFP-PODXL and RFP-NHERF1 patterns are similar between days 4 and 8, but few cilia are present on day 4. Bar, 20 µm. (C) Live cells expressing GFP-PODXL were imaged and individually scored for the appearance of a GFP-PODXL exclusion zone in the center of the apical membrane. Three fields of cells were counted for each of two experiments, and the percentage of cells with apparent exclusion zones appears on each image. On day 1, only 16/150 cells appeared to have a GFP-PODXL exclusion zone. The fraction rose on day 2 (46/214) and dramatically increased on day 3 (166/257) before reaching a plateau on day 4 (182/250). Images were collected with identical microscope settings. Bar, 10 µm. (D) 24 h after seeding on a filter, cells were fixed and stained with anti–γ-tubulin to visualize centrioles and anti-gp135 to visualize endogenous podocalyxin. Centrioles are aligned below the center of the apical membrane before podocalyxin is excluded from the CMD. Small podocalyxin exclusion zones are only seen in fixed samples. Images represent a single confocal plane. Bar, 20 µm. (E) SEM imaging of cells 4 d after seeding surprisingly shows microvilli in the CMD. After 8 d, an area at the base of primary cilia (arrowheads) is free of microvilli. Images in A–C are projected z stacks. Bars: (left) 5 µm; (right) 2 µm.

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