Only a small number of Atg9 vesicles are involved in a single round of autophagosome formation. (A) Wild-type cells expressing both Atg9-2×GFP and Atg17-2×mCherry were starved for 2 h and observed at 30 ms/frame. GFP fluorescence intensities of cytoplasmic mobile Atg9 vesicles and PAS-assembled Atg9 vesicles were quantitated as in Fig. 2 J. Error bars indicate standard deviation. Arrowheads indicate Atg9-2×GFP vesicles and Atg17-2×mCherry assembling at the PAS. a.u., arbitrary unit; Stv., starved. (B) FRAP analysis of Atg proteins at the PAS. ATG9-2×GFP mRFP-APE1 cells and GFP-ATG8 mRFP-APE1 cells were treated with rapamycin for 30 min and then subjected to FRAP analysis. Arrowheads indicate positions of photobleaching. (C) Quantitation of FRAP observations in B; normalized relative fluorescence intensities (RFI) after photobleaching of Atg9-2×GFP (n = 9) and GFP-Atg8 (n = 10). Error bars indicate standard deviation. (D) ypt7Δ atg11Δ cells and ypt7Δ atg11Δ atg17Δ cells expressing Atg9-2×GFP were starved for 3 h and observed at 30 ms/frame (see also Video 10). Arrows and arrowheads indicate cytoplasmic Atg9-2×GFP vesicles and Atg9-2×GFP clusters located on the autophagosomal membranes, respectively. (E) Multiple Atg9 vesicles are mixed during autophagy. Wild-type cells and atg11Δ atg17Δ cells expressing Atg9-2×Kaede were treated with rapamycin for 1 h. After UV irradiation (0 h), the cells were subjected to prolonged chase incubation for 2, 5, or 6 h. Arrowheads indicate Atg9-2×Kaede vesicles observed with both red and green fluorescence. (F) The number of mobile Atg9-2×Kaede vesicles observed with either red fluorescence alone or with both red and green fluorescence in E were counted. To exclude Atg9 signals colocalized at the PAS, immobile Atg9 puncta were not counted. The data shown are from a single representative experiment out of three repeats. WT, wild type. Bars: (A, D, and E) 5 µm; (B) 3 µm.