Figure 1

Establishment of the FLAD assay. (A) Schematic model of the FLAD assay. (B) Representative images of a cell subjected to the FLAD assay. Wild-type MEFs transiently expressing TagRFP were treated with 0.5 \(\upmu \)M rapamycin for 3 h to induce canonical autophagy. Then, the FLAD assay was performed. Image 1: just before photobleaching. Image 2: during photobleaching. Half of the cell area in the confocal plane (green rectangle) was photobleached. Image 3: just after photobleaching. Green and blue circle: region used to measure fluorescence intensity of the bleached area and non-bleached area, respectively. The TagRFP fluorescence disappeared only in the bleached area. Image 4: 108 s after photobleaching. Several FLAD⁺ puncta were observed as bright TagRFP puncta (arrowheads) in the non-bleached area. The ROI is shown by the yellow dotted rectangle and the magnified image is shown in the inset. Bars = 10 \(\upmu \)m. (C) Time-dependent alterations in fluorescence intensity in the bleached area (green circle in image 3 of B), the non-bleached area (blue circle in image 3 of B), and FLAD⁺ puncta (yellow dotted circle in image 4 of B) was monitored continuously. Note that the blue and green circles are almost an equal distance from the borderline of the bleached area. (D) Equilibration time of wild-type MEFs treated with or without rapamycin (0.5 µM). Cytoplasmic intensities were equilibrated at 91.1 ± 50.6 s in control cells (n = 5) and 97.4 ± 53.1 s in cells treated with rapamycin for 3 h (n = 4). Error bars indicate the S.D. Statistical analysis was performed by the Student t-test. ns: no significant difference.