Calculation of diffusion coefficients and escape times for PIN2. (A) Distribution of diffusion coefficients (D) for PIN2-EosFP on the plasma membrane of fresh protoplasts (n = 648 tracks from four protoplasts) in a histogram with logarithmically spaced bins. The data were fitted using a Gaussian distribution (red curve). Single-particle tracks of PIN2-EosFP on the plasma membrane of protoplasts were visualized by TIRFM and used to calculate MSD. D values were calculated by linear fit to MSD versus time. (B) Quantitative FRAP experiments of 16 protoplasts to determine diffusion coefficient of PIN2-GFP. Fluorescence intensities during recovery after photobleaching are plotted versus time. (C) Electron tomography slice of a single ILV bud from an Arabidopsis root cell. The yellow line indicates the position of the contour used to segment the ILV bud in IMOD. Bar, 10 nm. (D) Extracted 3D geometry from the segmented ILV bud shown in C and used to run the simulations shown in E–G. Green indicates the lower ILV hemisphere, orange indicates the ILV bud neck, and blue indicates the rest of the ILV bud membrane. Numbers shown in nanometers. (E–G) Simulated escape times of 40 PIN2 particles placed on the lower hemisphere of the ILV bud using the two D values experimentally calculated by TIRFM (0.06 µm²) and by FRAP (0.17 µm²). The colored lines indicate the mean numbers of remaining cargo over time calculated from >300 simulations, and the shaded regions correspond with the SD. Gray vertical lines indicate the mean time required for 50% of the PIN2 particle to escape the ILV bud. Simulations were run either without (E) or with (F) volume exclusion and with a fivefold decrease in D when PIN2 particles entered the neck region (G). VE, volume exclusion.