Deterministic Early Endosomal Maturations Emerge From a Stochastic Trigger-and-Convert Mechanism

Endosomal maturation is critical for robust and timely cargo transport to specific cellular compartments. The most prominent model of early endosomal maturation involves phosphoinositide-driven gain or loss of specific proteins on individual endosomes, emphasising an autonomous and stochastic description. However, limitations in fast, volumetric imaging long hindered direct whole-cell measurements of absolute numbers of maturation events. Here, we use lattice light-sheet imaging and bespoke automated analysis to track individual very early (APPL1-positive) and early (EEA1-positive) endosomes over the entire population, demonstrating that direct interendosomal contact drives maturation. Using fluorescence lifetime, we show that this interaction is underpinned by asymmetric EEA1 binding to very early and early endosomes through its N- and C-termini, respectively. In combination with agent-based simulation that confirms a ‘trigger-and-convert’ model, our findings indicate that APPL1-to EEA1-positive maturation is driven not by autonomous events but by heterotypic EEA1-mediated interactions, providing a mechanism for temporal and population-level control of maturation. ### Competing Interest Statement The authors have declared no competing interest.