Department of Biomolecular Chemistry, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI 53706.
Department of Biomolecular Chemistry, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI 53706
Proc Natl Acad Sci U S A. 2019 Apr 2;116(14):6858-6867. doi: 10.1073/pnas.1817898116. Epub 2019 Mar 20.
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.
多泡体（MVEs）的形成介导了许多整合膜蛋白的周转，并且与生长因子信号的下调有关，因此表现出肿瘤抑制因子的特性。用于运输的内体分选复合物（ESCRT）机制在 MVE 的生物发生中起着关键作用，使货物选择和腔内小泡（ILV）出芽成为可能。然而，哺乳动物细胞中内源性 ESCRT 复合物组装和拆卸的时空模式仍然定义不明确。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，我们在多种生长条件下确定了 MVEs 中早期和晚期作用的 ESCRT 成分的天然动力学。具体而言，我们的数据表明，ESCRT-0 快速积聚在内体上，通常在 30 秒内，其水平的波动方式取决于 ESCRT-I 的下游募集。同样，ESCRT-I 复合物的水平也在内体上波动，但与 ESCRT-0 相比，其平均停留时间长 5 倍以上。然而，Vps4 的积累是最短暂的，这表明 ILV 形成的完成发生得非常快。在添加表皮生长因子（EGF）后，ESCRT-I 和 Vps4 都在 endosomes 上保留了很长一段时间，而 ESCRT-0 的动力学仅受到适度影响。我们的发现与一种模型一致，即生长因子刺激稳定了 ESCRT 机制的晚期作用成分在 endosomes 上，以加速 ILV 生物发生的速度，并减弱受体激活引发的信号转导。
