Figure 2. GFP-PODXLΔ4 and GFP-GPI are... | Rockefeller University Press

$Figure 2. GFP-PODXLΔ4 and GFP-GPI are found in the CMD and cilia of live MDCK cells, but fixation can create the appearance of CMD exclusion. (A) Live cells expressing GFP-PODXLΔ4 or GFP-GPI were imaged 4 d after seeding and, in contrast to GFP-PODXL, failed to show exclusion from the CMD. Images were collected with microscope settings identical to Fig. 1 C. GFP-PODXL day 4 image from Fig. 1 C is shown for comparison. (B) Paraformaldehyde-fixed cells expressing GFP-PODXL (top row) or GFP-PODXLΔ4 (bottom row) were stained with phalloidin 4 d after seeding. GFP-PODXLΔ4 appeared to be excluded from the CMD in many cells. (C) Cells expressing GFP-PODXL, GFP-PODXLΔ4, or GFP-GPI were imaged and individually scored for the appearance of an exclusion zone. In live cells, GFP-PODXL was excluded from the CMD in 182/250 cells scored, in contrast to GFP-PODXLΔ4 (5/276) and GFP-GPI (11/269). In fixed cells, GFP-PODXL was still excluded from the CMD in most cells (209/243), but the fraction of cells that also excluded GFP-PODXLΔ4 increased from <5 to 40% (82/204). (D) Images of live cells 12 d after seeding on filters show that GFP-PODXLΔ4 and GFP-GPI are present in both the CMD and ciliary membrane. (top row) Z stacks of the apical membrane of live cells expressing GFP-PODXL, GFP-PODXLΔ4, or GFP-GPI were projected into a single image. Images were collected with identical microscope settings to Fig. 1 C, but at lower digital zoom. (bottom row) Single confocal sections above the apical membrane were taken from the z stacks in the top row, and image brightness was increased. Visible cilia are labeled with arrowheads. (E) Cells expressing GFP-PODXL, GFP-PODXLΔ4, or GFP-GPI (green) were fixed and stained with antiacetylated tubulin (white) to image primary cilia 12 d after seeding on filters. (F) Cilia longer than 2 µm extending from GFP-positive cells were counted in five fields in each of two experiments. In fixed cells, cilia were identified by antiacetylated tubulin (ciliated); in live cells, cilia were identified as GFP-positive projections from the center of the apical membrane (GFP + cilium). Scoring GFP-PODXL–expressing cells, 54/188 fixed cells were ciliated, but only 4/144 live cells had a GFP-positive cilium. In contrast, GFP-PODXLΔ4–expressing cells had similar numbers of cilia that could be detected using the two methods (24/77 fixed and 34/126 live), as did GFP-GPI–expressing cells (77/219 fixed and 64/194 live). Images in A, B, and E are projections of z stacks. Error bars represent standard deviation between experiments. Bars, 10 µm.$

Figure 2.

GFP-PODXLΔ4 and GFP-GPI are found in the CMD and cilia of live MDCK cells, but fixation can create the appearance of CMD exclusion. (A) Live cells expressing GFP-PODXLΔ4 or GFP-GPI were imaged 4 d after seeding and, in contrast to GFP-PODXL, failed to show exclusion from the CMD. Images were collected with microscope settings identical to Fig. 1 C. GFP-PODXL day 4 image from Fig. 1 C is shown for comparison. (B) Paraformaldehyde-fixed cells expressing GFP-PODXL (top row) or GFP-PODXLΔ4 (bottom row) were stained with phalloidin 4 d after seeding. GFP-PODXLΔ4 appeared to be excluded from the CMD in many cells. (C) Cells expressing GFP-PODXL, GFP-PODXLΔ4, or GFP-GPI were imaged and individually scored for the appearance of an exclusion zone. In live cells, GFP-PODXL was excluded from the CMD in 182/250 cells scored, in contrast to GFP-PODXLΔ4 (5/276) and GFP-GPI (11/269). In fixed cells, GFP-PODXL was still excluded from the CMD in most cells (209/243), but the fraction of cells that also excluded GFP-PODXLΔ4 increased from <5 to 40% (82/204). (D) Images of live cells 12 d after seeding on filters show that GFP-PODXLΔ4 and GFP-GPI are present in both the CMD and ciliary membrane. (top row) Z stacks of the apical membrane of live cells expressing GFP-PODXL, GFP-PODXLΔ4, or GFP-GPI were projected into a single image. Images were collected with identical microscope settings to Fig. 1 C, but at lower digital zoom. (bottom row) Single confocal sections above the apical membrane were taken from the z stacks in the top row, and image brightness was increased. Visible cilia are labeled with arrowheads. (E) Cells expressing GFP-PODXL, GFP-PODXLΔ4, or GFP-GPI (green) were fixed and stained with antiacetylated tubulin (white) to image primary cilia 12 d after seeding on filters. (F) Cilia longer than 2 µm extending from GFP-positive cells were counted in five fields in each of two experiments. In fixed cells, cilia were identified by antiacetylated tubulin (ciliated); in live cells, cilia were identified as GFP-positive projections from the center of the apical membrane (GFP + cilium). Scoring GFP-PODXL–expressing cells, 54/188 fixed cells were ciliated, but only 4/144 live cells had a GFP-positive cilium. In contrast, GFP-PODXLΔ4–expressing cells had similar numbers of cilia that could be detected using the two methods (24/77 fixed and 34/126 live), as did GFP-GPI–expressing cells (77/219 fixed and 64/194 live). Images in A, B, and E are projections of z stacks. Error bars represent standard deviation between experiments. Bars, 10 µm.

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