Simulation of cargo escape in concatenated ILV geometries. (A) Geometries derived from electron tomograms of plant MVEs used for the cargo diffusion simulations depicted in B. Green indicates the lower ILV hemisphere, where the PIN2 molecules were placed to start the simulation, orange indicates the ILV bud neck and interconnecting membrane bridges, blue indicates the rest of the internalized membrane, and white indicates the endosomal limiting membrane. Bars, 10 nm. (B) Simulated escape times of 40 PIN2 particles placed on the lower hemisphere of the ILV bud using two D values (0.06 and 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 times required for 50% of the PIN2 particle to escape the ILV bud. Simulations were run incorporating volume exclusion and a fivefold decrease in D when PIN2 particles entered neck or bridge regions (orange in A).