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First published online 15 January 2003
doi: 10.1242/jcs.00289

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Real-time analysis of clathrin-mediated endocytosis during cell migration

The Laboratory of Cellular Biophysics, The Rockefeller University, 1230 York Avenue, Box 304, New York, NY 10021, USA

View larger version (50K): [in a new window]   Fig. 1. Analysis of clathrin dynamics in a migrating cell by TIR-FM. (A) A TIR-FM still image (from a sequence taken at 300 milliseconds per frame) demonstrating the presence of numerous dsRed-clathrin basal plasma membrane associated puncta. Scale bar, 5 µm. (B) 12 sequential images taken from an image stream of 200, enlarging region 1 from A. The frames depict the lateral movement of the clathrin punctum marked by the arrowhead. (C) 12 sequential images enlarging region 2 from A. These images depict the disappearance of the clathrin punctum marked by the arrowhead, presumably via internalization. (D) 18 sequential images enlarging region 3 from A, demonstrating the increasing fluorescence at the spot marked by the arrowhead. (E) Quantification of the fluorescence associated with a single disappearing dsRed-clathrin punctum.

View larger version (53K): [in a new window]   Fig. 2. Colocalization of dsRed-clathrin in Alexafluor488-transferrin puncta. (A) Alexafluor488-transferrin TIR-FM image. (B) dsRed-clathrin TIR-FM image of the same cell as in A. Spots marked by arrows contain both transferrin and clathrin. Scale bar, 5 µm. (C) The decrease of mean ± s.e.m. of correlation coefficient comparing images in A and B following pixel shift in all four directions. (D) Bar graph comparing the average clathrin fluorescence per unit area ± s.e.m. in regions within 150 transferrin puncta (50 per cell) to an equivalent number of regions outside of transferrin spots.

View larger version (48K): [in a new window]   Fig. 3. Colocalization of dynamin2-EGFP in dsRed-clathrin puncta in migrating MDCK cells. (A) dsRed-clathrin epifluorescence image. (B) Dynamin2-EGFP epifluorescence image. (C) Overlay of A and B. Scale bar, 5 µm. (D) dsRed-clathrin TIR-FM image. (E) Dynamin2-EGFP TIR-FM image. (F) Overlay of D and E. Scale bars, 5 µm. (G) Depiction of the decrease of mean ± s.e.m. of correlation coefficient comparing images in D and E following pixel shift in all four directions. (H) Graph of the dynamin2-EGFP fluorescence relative to dsRed-clathrin fluorescence (normalized to whole cell relative fluorophore intensity for each cell) within 150 clathrin puncta from three cells (50 per cell).

View larger version (69K): [in a new window]   Fig. 4. Colocalization of dynamin2-EGFP and dynamin1-EGFP in disappearing dsRed-clathrin puncta. (A) dsRed-clathrin TIR-FM image. Scale bar, 5 µm. (B) Dynamin2-EGFP TIR-FM image. (C) Five sequential images (300 milliseconds per frame) enlarging area outlined in A. (D) Five sequential images enlarging area outlined in B. (E) Graphic depiction of the disappearance of eight dsRed-clathrin and dynamin2-EGFP containing spots; values are presented as the mean ± s.e.m. of the average fluorescence per unit area relative to maximum value obtained for each spot minus the minimum value for each. (F, G) Five images (300 milliseconds per frame) demonstrating the behavior of dsRed-clathrin (F) and dynamin1-EGFP (G) prior to and during internalization. Images depicted represent frames 0, 50, 100, 150 and 200. (H) Graphic depiction of the disappearance of eight dsRed-clathrin and dynamin1-EGFP containing spots; values are presented as the mean ± s.e.m. of the average fluorescence per unit area relative to maximum value obtained for each spot minus the minimum value for each. Values depicted were taken from an aligned data set of 40 seconds. In E and H, the fluorescence traces of each of the eight spots evaluated were temporally aligned to the start of dynamin1/2-EGFP fluorescence decrease.

View larger version (34K): [in a new window]   Fig. 5. Migration of MDCK cells transfected with dsRed-clathrin. Cells were imaged via time-lapse epifluorescence microscopy migrating towards the area of monolayer wounding (the top of the field). This figure presents the first (A) and last (B) images of a 1 hour time-lapse sequence, and an overlay (C) of cell borders drawn to illustrate net cell displacement. Scale bar, 20 µm.

View larger version (92K): [in a new window]   Fig. 6. The distribution of plasma-membrane-associated dsRed-clathrin and dynamin2-EGFP in migrating cells. (A) Epifluorescence image of a migrating MDCK cell expressing dsRed-clathrin. (B) TIR-FM image of the same cell overlying the cell border drawn in A, and depicting the apparent migratory direction of this cell in addition to three regions along this trajectory. (C) Epifluorescence image of a migrating MDCK cell expressing dynamin2-EGFP. (D) TIR-FM image of the same cell overlying the cell border drawn in C, and depicting the apparent migratory direction of this cell in addition to three regions along this trajectory. Scale bar, 5 µm.

View larger version (13K): [in a new window]   Fig. 7. Polarized distribution of markers for clathrin-mediated endocytosis in the basal membrane of migrating cells. (A) The quantification of the relative dsRed-clathrin fluorescence intensity per unit area within three regions along the migrating axis of the cell. The average of seven cells ± s.e.m. is plotted. (B) The quantification of the relative dynamin2-EGFP fluorescence intensity per unit area within three regions along the migrating axis of the cell. The average of six cells ± s.e.m. is plotted. (C) The quantification of the relative Alexafluor488-transferrin fluorescence intensity per unit area within three regions along the migrating axis of the cell. The average of four cells ± s.e.m. is plotted.

View larger version (43K): [in a new window]   Fig. 8. Distribution of clathrin-mediated endocytosis in the basal plasma membrane of migrating cells. (A) TIR-FM image of the basal plasma membrane ECFP-Mem distribution in a migrating MDCK cell co-transfected with dsRed-clathrin and ECFP-Mem. Cell border was drawn around the entire region of fluorescent signal. (B) dsRed-clathrin TIR-FM image of the same cell overlying the cell border drawn in A. The cell is apparently migrating in the direction denoted by the arrow. (C) The relative dsRed-clathrin fluorescence intensity per unit area divided by the relative ECFP-Mem fluorescence intensity per unit area within three regions of the migrating cell. The average of six cells ± s.e.m. is plotted. (D) The quantification of the relative dsRed-clathrin spot number per unit area within the three cell regions. The average ± s.e.m. is plotted (three cells, 1507 spots). (E) The quantification of the relative dsRed-clathrin spot disappearance per unit area within the three cell regions. The average ± s.e.m. is plotted (three cells, 237 spots).

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