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

Abstract

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. lattice light sheet microscopy | ESCRT microdomain | CRISPR/Cas9 | epidermal growth factor receptor | organelle

Exosome-Derived LncRNAs in Lung Cancer

“…ILVs fused with the serous membrane to form multivesicular endosomes (MVEs) with packaging various bioactive molecules. Endosomal sorting complex required for transport (ESCRT) is the key molecular mechanism in MVE membrane shaping and scissing, and as a primary driver, ESCRT enables cargo selection and ILV budding ( 55 – 57 ). Mediated by Rab family and soluble N -ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) family, mature exosomes are released by donor cells ( 58 , 59 ).…”

Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions

“…A serious problem with this approach, however, is that exogenous expression of FP-tagged ESCRT subunits by transfection or transduction generally yields excess subunits (overexpression), which can lead to dominant negative artifacts and ESCRT dysfunction (39, 40). The presence of the untagged native ESCRT protein in the cells also makes it uncertain whether the behavior of an overexpressed FP-ESCRT probe accurately reflects the activity of the native protein (41). Indeed, dynamics of overexpressed FP-ESCRT probes have been shown in some cases to differ significantly from the dynamics of the endogenous ESCRTs (41).…”

“…The presence of the untagged native ESCRT protein in the cells also makes it uncertain whether the behavior of an overexpressed FP-ESCRT probe accurately reflects the activity of the native protein (41). Indeed, dynamics of overexpressed FP-ESCRT probes have been shown in some cases to differ significantly from the dynamics of the endogenous ESCRTs (41). Using immunofluorescence to directly detect endogenous ESCRT subunits avoids these problems, but unfortunately, commercially available antibodies reliable enough for sensitive applications such as superresolution imaging are not available for most of the ESCRT proteins (33, 42, 43), and this approach also has the disadvantage of requiring fixation and permeabilization, which sacrifices dynamic information that can be obtained from living cells and can cause additional artifacts (44).…”

Connecting the dots: combined control of endocytic recycling and degradation

“…This perspective was solidified with the discovery of the ESCRT complex, a mechanistic view of transmembrane protein sorting centered around active sorting towards degradation emerged. The ESCRT complex is a large multiprotein complex which is composed of four sub complexes (ESCRT-0, ESCRT-1, ESCRT-2 and ESCRT-3) [66][67][68]. ESCRT functions by capturing ubiquitylated cargo at the endosome and sorting them into intra luminal vesicles (ILVs) of multivesicular bodies (MVBs) [69].…”