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Fig. 9. Imaging of GFP-Rab5c, Tf and EGF. (A-C) COS-7 cells expressing GFP-Rab5c were exposed to Alexa-Fluor-568-Tf for 3 minutes followed by a wash and addition of unlabeled Tf at a concentration of 200 µg/ml. Cells were imaged continuously by TIRF-M alternating with epifluorescence as described above. After 30 minutes, when the vast majority of the Tf signal had disappeared from the cell, Alexa-Fluor-568-EGF was added, and imaging resumed. (A) Optical sections through the cell in the GFP channel, illustrating the distribution of Rab5 in the three-dimensional volume of the cell. Arrows point to the redistribution of Rab5 to the cell periphery in response to EGF. (B) The mean intensity of Rab5 in the peripheral and juxtanuclear regions over time after exposure to EGF was quantified in five independent cells. (C) TIRF images of the fluorescent ligands superimposed on the Rab5 image. Arrows point to regions of colocalization. (D) Colocalization between Rab5-GFP and Alexa-Fluor-568-EGF or Alexa-Fluor-568-Tf over time. Plotted are the percentage of Rab5 colocalized with each ligand in pixel-dense regions (two to three regions per cell) over the indicated time intervals. Values are mean ± s.e.m. of three independent experiments. Statistical significance was calculated from two-tailed paired Student's t-tests. *P<0.001. (E) Sequence of images of a cell expressing GFP-Rab5 and exposed to Alexa-Fluor-568-EGF for the times shown in each panel. Arrows point to regions containing Alexa-Fluor-568-EGF, which acquire GFP-Rab5. Similar results were observed in a minimum of five independent experiments.
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