Atg9-containing structures are observed by high temporal resolution microscopy and analyzed by single-particle tracking. (A) ATG9-2×GFP cells were treated with rapamycin for 2 h and observed by fluorescence microscopy at 20 ms/frame (see also Video 1). Magnified view of the boxed area is shown. (B) ATG9-2×GFP atg11Δ atg17Δ cells were treated with rapamycin for 1 h and observed at 30 ms/frame. Anp1-mCherry (Golgi), Nhx1-mCherry (endosome), and Idh1-mCherry (mitochondria) were used as organelle markers. Green fluorescence and red fluorescence were acquired concurrently. (C) ATG9-2×GFP atg11Δ atg17Δ cells were observed at 16 ms/frame and subjected to single-particle tracking analysis. (D) Trajectories of the Atg9 puncta indicated in C. (E) 20 examples of the mean square displacement (MSD) curves calculated from traces of Atg9 puncta. (F) Histograms of the diffusion coefficients of the Atg9 puncta observed in cells grown to logarithmic phase (nutrient [Nut.]) or cells starved for 2 h (Stv.). The histograms were fitted to Gaussian distributions; medians of the fitting curves are indicated. The data shown are from a single representative experiment out of two repeats. (G) ATG9-2×GFP ABP140-mCherry cells were treated with 100 µg/ml latrunculin A (LatA) for 20 min and observed by fluorescence microscopy at 32 ms/frame (see also Video 2).