Growth factor stimulation promotes multivesicular endosome biogenesis by prolonging recruitment of the late-acting ESCRT machinery.
Growth factor stimulation promotes multivesicular endosome biogenesis by prolonging recruitment of the late-acting ESCRT machinery.
复制标题
生长因子刺激通过延长晚效 ESCRT 机制的募集来促进多囊泡内体生物发生。
DOI：
10.1073/pnas.1817898116
复制
发表时间：
2019
影响因子：
11.1
通讯作者：
Audhya,Anjon
中科院分区：
文献类型：
--
作者：
Quinney,KyleB;Frankel,ElisaB;Shankar,Raakhee;Kasberg,William;Luong,Peter;Audhya,Anjon
多囊泡内皮体(MVEs)的形成介导了许多完整的膜蛋白的周转，并参与了生长因子信号的下调，从而显示出肿瘤抑制因子的特性。运输所需的内体分选复合体(ESCRT)机械在MVE的生物发生中发挥关键作用，使货物能够选择和腔内小泡(ILV)萌发。然而，内源性ESCRT复合体在哺乳动物细胞中的组装和拆解的时空模式仍然不清楚。通过结合CRISPR/Cas9介导的基因组编辑和使用晶格光片显微镜(LLSM)的活细胞成像，我们确定了多种生长条件下血管内皮细胞ESCRT早期和晚期作用的自然动态。具体地说，我们的数据表明ESCRT-0在内小体上快速积累，通常不到30秒，其水平以一种依赖于ESCRT-I下游招募的方式振荡。同样，ESCRT-I复合体的水平也会随着内体的变化而波动，但其平均停留时间比ESCRT-0短五倍以上。然而，Vps4的积累是最短暂的，这表明ILV的形成很快就会完成。在加入表皮生长因子(EGF)后，ESCRT-I和VPS4都在内体保留了相当长的一段时间，而ESCRT-0的动力学只受到轻微的影响。我们的发现与一个模型是一致的，在该模型中，生长因子刺激稳定内体ESCRT机制的晚期作用组件，以加快ILV的生物发生速度，并减弱由受体激活启动的信号转导。
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.