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Endosomal maturation is a critical and fundamental process for robust transport of cargo (such as activated receptors) to specific cellular compartments in a timely manner. The most prominent model of endosomal maturation involves a phosphoinositide-driven gain or loss of specific proteins at the level of individual endosomes, emphasising an autonomous and stochastic model of maturation. However, to date, direct whole cell-level measurements of absolute number of maturation events have not been performed, owing to limitations in fast, volumetric imaging. Here, we use lattice light-sheet imaging to track individual very early and early endosomes over the entire population. We demonstrate that direct inter-endosomal contact drives the maturation from very early (APPL1-positive) to early (EEA1-positive) endosomes. Using fluorescence lifetime, we show that this endosomal interaction is underpinned by the asymmetric binding of EEA1 to very early and early endosomes through the N- and C-termini, respectively. Thus, stochastic microtubule-mediated inter-endosomal interactions through EEA1 provide a mechanism to bring temporal and population-level control to the process of endosome maturation. Our findings indicate that APPL1- to EEA1-positive endosomal maturation is not a result of autonomous endosomal events but is driven by heterotypic EEA1-mediated endosomal interactions.

Multicellular life processes such as proliferation and differentiation depend on cell surface signaling receptors that bind ligands generally referred to as growth factors. Recently, it has emerged that the endosomal system provides rich signal processing capabilities for responses elicited by these factors [1-3]. At the single cell level, endosomal trafficking becomes a critical component of signal processing, as exemplified by the epidermal growth factor (EGF) receptors of the receptor tyrosine kinase family. EGFRs, once activated by EGF, are robustly trafficked to the phosphatase-enriched peri-nuclear region (PNR), where they are dephosphorylated [4-8]. However, the details of the mechanisms regulating the movements of stimulated EGFR in time and space, i.e., towards the PNR, are not known. What endosomal regulators provide specificity to EGFR? Do modifications to the receptor upon stimulation regulate its trafficking? To understand the events leading to EGFR translocation, and especially the early endosomal dynamics that immediately follow EGFR internalization, requires the real-time, long-term, whole-cell imaging of multiple elements. Here, exploiting the advantages of lattice light-sheet microscopy [9], we show that the binding of EGF by its receptor, EGFR, triggers a transient calcium increase that peaks by 30 s, causing the desorption of APPL1 from pre-existing endosomes within one minute, the rebinding of liberated APPL1 to EGFR within three minutes, and the dynein-dependent translocation of APPL1-EGF-bearing endosomes to the PNR within five minutes. The novel, cell spanning, fast acting network that we reveal integrates a cascade of events dedicated to the cohort movement of activated EGFR receptors. Our findings support the intriguing proposal that certain endosomal pathways have shed some of the stochastic strategies of traditional trafficking, and have evolved behaviors whose predictability is better suited to signaling [10, 11]. Work presented here demonstrates that our whole cell imaging approach can be a powerful tool in revealing critical transient interactions in key cellular processes such as receptor trafficking.