Effects of Paclitaxel on EGFR Endocytic Trafficking Revealed Using Quantum Dot Tracking in Single Cells
Figure 2
PTX restricts endocytic transports near the periphery of the cytoplasm.
(A) The time-lapse images of endocytic trafficking in A549 cells at t = 0, 5, 30, 60 min (time resolution: 100 ms, time stamp in min: sec). The fluorescent images of the QDs are overlaid with the bright-field DIC images of the cell. Endocytic vesicles moved toward the center of the cell and fused with other EVs continuously when traveling in the cytoplasm in the control cells. The EVs in the PTX-treated cells remained near the periphery and fused with other EVs in a minor fashion. The concentration of PTX used is 100 nM. See Movies S2 and S3. (B) Illustration of the endocytic ratio calculation. The intracellular QDs are marked by red stars, whereas extracellular ones are marked by green stars. The boundaries and nuclear of the cell are highlighted in yellow and blue lines, respectively. The center of the cell is marked by a green dot in the nucleus. All the sites are selected in DIC images. The change of the cell shapes in 60 min is negligible, as examined from the DIC images obtained before and after every 30 min fluorescence imaging. The distance of QDs from the center of the cell is denoted by the solid arrow ‘a’, and the distance of the corresponding points on the cell boundaries to the center of the cell is denoted by the hollow arrow ‘b’. The endocytic ratio equals the mean value of b/a. (C) The endocytic ratio of the control and PTX-treated cells in panel A is a function of time. Red lines represent the smooth data obtained by averaging 20 adjacent points along the original lines. (D) The merged fluorescent images of QD (red) and MTs (green) after 60 min of EGF-QD endocytosis under the identical condition to panel A. Cells were labeled with rat anti-Tubulin and Alexa-488 goat anti-rat IgG conjugate. The nucleus was stained with DAPI (blue). All scale bars: 10 .