AMPK regulates the tethering of WIPI2-positive phagophores. (A) Model depicting the diffusion properties of tethered phagophores and untethered ATG9 vesicles. (B and C) Step size distributions of Halo-ATG13, ULK1-Halo, Halo-WIPI2, and Halo-ATG2A trajectories for (B) LC3B-positive and (C) LC3B-negative autophagy factor foci. (D) Kernel density plots of the step size distributions of Halo-ATG13, ULK1-Halo, Halo-WIPI2, and Halo-ATG2A trajectories for LC3B-positive and -negative tracks in the indicated media conditions. The vertical white solid line represents the median of step size distribution. (E) Step size distributions of LC3B-positive and -negative Halo-WIPI2 trajectories fit with a two-state diffusion model (black line) encompassing tethered (red) and untethered (blue) populations in the different media conditions. (F) Diffusion coefficients of the tethered and untethered populations of LC3B-positive and -negative Halo-WIPI2 trajectories in the different media conditions, derived from the fits shown in E. (G) Distribution of LC3B-positive and -negative Halo-WIPI2 trajectories between tethered and untethered populations, derived from the fits shown in E. Statistical significance was assessed using a two-tailed t-test (*P < 0.05, **P < 0.01).