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First published online August 24, 2006
doi: 10.1242/10.1242/jcs.03066

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Decreased polarity and increased random motility in PtK1 epithelial cells correlate with inhibition of endosomal recycling

¹ The Burnham Institute, 10901 N. Torrey Pines Road, Room 7108, La Jolla, CA 92037, USA
² The Scripps Research Institute, Department of Cell Biology, CB163, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA

View larger version (41K): [in a new window]   Fig. 1. Recycling in a cell at the edge of epithelial island. AJ, adherens junctions; CCP, clathrin-coated pits; EE, early/sorting endosomes; ERC, endosomal recycling compartment; FA, focal adhesions; LE, late endosomes. Relevant Rab players are indicated (Rab4, Rab5 and Rab11).

View larger version (120K): [in a new window]   Fig. 2. GFPRab11b localization in stably expressing PtK1 cell lines. Confocal images of living PtK1 cells expressing GFPRab11b-GDP (A,D,G) and GFPRab11b-GTP (B,E,H) visualized by GFP fluorescence. Alexa-568-transferrin (red) in control cells (C,F) and in cells stably expressing the GFPRab11b mutants (D,E,G,H). Live cell images were taken approximately 5 minutes (C-E) or 30 minutes (F-H) after transferrin internalization was induced by a shift from 0°C to 37°C. Corresponding supplemental movies are available for control (supplementary material Movie 1a,b), GFPRab11b-GDP (supplementary material Movie 2a,b) and GFPRab11b-GTP (supplementary material Movie 3a,b) cells.

View larger version (74K): [in a new window]   Fig. 3. Rab11b-GFP localization in transiently expressing cells. A live GFPRab11b-GDP-expressing cell showing partial colocalization of the GFPRab11b-GDP (A) with Alexa-568-transferrin internalized under steady-state conditions (B). (C) GFPRab11b-GDP (green), Alexa 568 transferrin (red). Colocalization (G, white) between GFPRab11b-GDP (D,G, green), antibodies against Rab11 (E,G, blue) and Alexa-568-transferrin (F,G, red).

View larger version (54K): [in a new window]   Fig. 4. Transferrin recycling is inhibited in cells expressing GFPRab11b mutants. (A,B) Binding (on ice) of Alexa-568-transferrin to control and to cells transiently expressing GFPRab11b-GDP. (C,D) Transferrin retained in the GFPRab11b-GDP-expressing cell after a 30 minute chase at 37°C. The outlines of individual cells in the island are indicated by dotted lines. (A,C) GFPRab11b-GDP-expressing cells identified by GFP fluorescence; (B,D) Alexa-568-transferrin. (E) An assay for specificity of transferrin binding and uptake by PtK1 cells. Cells were incubated with increasing concentrations (10, 50, 100 and 250 µg/ml each) of fluorescently labeled Alexa568-transferrin and FITC-dextran either at 37°C or on ice. Each bar represents average fluorescence intensity of approximately 100 cells per condition. Each condition was assayed in three different wells, four fields of view per well and analyzed using Thora software. (F) Transferrin recycling in cells transiently expressing GFPRab11b-GDP, measured as fluorescent intensity of Alexa-568-transferrin retained inside the cells at the end of a 30-minute chase period, and expressed as a percentage of total transferrin bound at the beginning of experiment. (G) Recycling efficiency in cells transiently expressing GFPRab11b-GDP, expressed as a percentage of neighboring control cells values. All data are mean ± s.e.m., calculated from at least three experiments, 20-40 cells per experiment. (H) Correlation between the GFPRab11b-GDP expression level in a stable GFPRab11b-GDP cell line and transferrin retained inside the cells after a 1 hour recycling period. Data for 40 cells from a representative experiment are shown.

View larger version (73K): [in a new window]   Fig. 5. Cells transiently expressing GFPRab11b mutants exhibit abnormal motility and morphology. The cells had been microinjected with GFPRab11b constructs approximately 4 hours before the beginning of the time-lapse, and expressing cells are marked with white dots. Frames are 40 minutes apart. (A) control PtK1 island (see also supplementary material Movie 4) and islands in which some of the cells were microinjected with either the GFPRab11b-GTP (B, supplementary material Movie 5) or GFPRab11b-GDP (C, supplementary material Movie 6) construct. The expressing cells are marked with a white dot. In C, the cells expressing high levels of GFPRab11b-GDP are marked with arrows in the first image and white dots thereafter; the unmarked cell expresses very low levels of GFPRab11b-GDP. (D-F) Tracks of migration of control cells (D) and cells expressing GFPRab11b-GTP (E) or GFPRab11b-GDP (F). Tracks were generated from 6 hour, 4-minutes interval time-lapse movies of cellular islands shown in A-C. Start positions of the cells are marked by gray circles.

View larger version (104K): [in a new window]   Fig. 6. Cells expressing GFPRab11b mutants are not able to migrate efficiently in an experimental wound assay due to a directionality defect. Confluent cell monolayers were scratch-wounded to induce migration into the wound. Positions of cells approximately 60 minutes after wounding the monolayers (`start', A,D,G,J) and 6 hours later (`finish', B,E,H,K) are shown for the control PtK1 cells (A,B), cells stably expressing GFPRab11b-GDP (D,E) and GFPRab11b-GTP (G,H). The tracks of individual cells at the wound edges, as determined from time-lapse movies, are indicated in the `start' micrographs (A,D,G). Each frame constitutes a wound segment of approximately 400 µm in length. Graphs in panels C, F and I show X and Y positions of individual cells at the wound edge for the mutants in the micrographs above (the starting points of all cells are placed at 0:0). Data points are 2 hours apart. (J) Average translocation of the edge (i.e. total advancement into the wound) in 6 hours. (K) Mean average velocities of the mutant Rab11b-expressing cells at the wound edges and in the middle of the monolayers. Corresponding supplemental movies are available for control (supplementary material Movie 7), GFPRab11b-GDP (supplementary material Movie 8) and GFPRab11b-GTP (supplementary material Movie 9) cells.

View larger version (103K): [in a new window]   Fig. 7. Effects of GFPRab11b-GDP expression on the cytoskeleton and on adhesion. (A) GFPRab11b-GDP-expressing cells identified by GFP fluorescence; actin (B) and microtubules (C) identified by immunofluorescence; image of the nuclei (blue) identified by DAPI is overlaid onto the microtubule image (C). (D-G) Cell-cell adhesions visualized by indirect immunofluorescence against e-cadherin. (D,F) Pseudocolored overlay images of GFPRab11b-GDP (green), E-cadherin (red) and nuclei (blue); (E,G) E-cadherin images of the same cells. (H,I) Confocal micrographs of focal adhesions visualized by indirect immunofluorescence against vinculin. (H) Pseudocolored overlay image of GFPRab11b-GDP (green) and vinculin (red); (I) vinculin image of the same cells. Scale bar for all panels, 20 µm.

View larger version (32K): [in a new window]   Fig. 8. Decreased recycling correlates with increased motility in PtK1 cells. (A) Transferrin recycling in cells transiently expressing various Rab mutants measured as fluorescent intensity of Alexa-568-transferrin retained inside the cells at the end of a 30-minute chase period, and expressed as a percentage of total transferrin bound at the beginning of experiment. (B) Recycling efficiency in cells transiently expressing various Rab mutants, expressed as a percentage of neighboring control cell values. (C) Velocities of cells transiently expressing various Rab mutants. (D) Correlation between cell velocity (as a percentage of control) and transferrin recycling (as a percentage of total transferrin bound at the beginning of the experiment). All data points are mean±s.e.m., calculated from at least three experiments, 20-40 cells per experiment.