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Growth factor stimulation promotes multivesicular endosome biogenesis by prolonging recruitment of the late-acting ESCRT machinery

Significance The formation of multivesicular endosomes (MVEs) enables the sequestration of numerous membrane-associated cargo molecules, including signaling receptors, to downregulate signal transduction pathways initiated by various stimuli. In this work, we demonstrate that the machinery responsible for MVE biogenesis responds to growth factor stimulation by extending its duration of action at endosomes. Specifically, we show that endosomal sorting complex required for transport-I (ESCRT-I) and Vps4 accumulation at MVEs is dramatically prolonged following growth factor stimulation. Additionally, we demonstrate that native ESCRT complexes exhibit an oscillatory behavior on endosomes, which is dependent on the sequential recruitment of downstream components. Our studies strongly suggest that components of the ESCRT machinery act cyclically and in a coordinated manner to enable a rapid response to changing environmental cues. The formation of multivesicular endosomes (MVEs) mediates the turnover of numerous integral membrane proteins and has been implicated in the down-regulation of growth factor signaling, thereby exhibiting properties of a tumor suppressor. The endosomal sorting complex required for transport (ESCRT) machinery plays a key role in MVE biogenesis, enabling cargo selection and intralumenal vesicle (ILV) budding. However, the spatiotemporal pattern of endogenous ESCRT complex assembly and disassembly in mammalian cells remains poorly defined. By combining CRISPR/Cas9-mediated genome editing and live cell imaging using lattice light sheet microscopy (LLSM), we determined the native dynamics of both early- and late-acting ESCRT components at MVEs under multiple growth conditions. Specifically, our data indicate that ESCRT-0 accumulates quickly on endosomes, typically in less than 30 seconds, and its levels oscillate in a manner dependent on the downstream recruitment of ESCRT-I. Similarly, levels of the ESCRT-I complex also fluctuate on endosomes, but its average residency time is more than fivefold shorter compared with ESCRT-0. Vps4 accumulation is the most transient, however, suggesting that the completion of ILV formation occurs rapidly. Upon addition of epidermal growth factor (EGF), both ESCRT-I and Vps4 are retained at endosomes for dramatically extended periods of time, while ESCRT-0 dynamics are only modestly affected. Our findings are consistent with a model in which growth factor stimulation stabilizes late-acting components of the ESCRT machinery at endosomes to accelerate the rate of ILV biogenesis and attenuate signal transduction initiated by receptor activation.