Center for Cell and Genome Science, University of Utah, Salt Lake City, Utah, United States of America.
Department of Biology, University of Utah, Salt Lake City, Utah, United States of America.
PLoS One. 2020 Sep 4;15(9):e0237268. doi: 10.1371/journal.pone.0237268. eCollection 2020.
Endosomal sorting complexes required for transport proteins (ESCRT) catalyze the fission of cellular membranes during budding of membrane away from the cytosol. Here we have used Total Internal Reflection Fluorescence (TIRF) microscopy to visualize the recruitment of ESCRTs specifically, ALIX, CHMP4b and VPS4 onto the budding HIV Gag virus-like particles (VLPs). We imaged the budding VLPs with 200 millisecond time resolution for 300 frames. Our data shows three phases for ESCRT dynamics: 1) recruitment in which subunits of ALIX, CHMP4b and VPS4 are recruited with constant proportions on the budding sites of HIV Gag virus like particles for nearly 10 seconds, followed by 2) disassembly of ALIX and CHMP4b while VPS4 signal remains constant for nearly 20 seconds followed by 3) disassembly of VPS4. We hypothesized that the disassembly observed in step 2 was catalyzed by VPS4 and powered by ATP hydrolysis. To test this hypothesis, we performed ATP depletion using (-) glucose medium, deoxyglucose and oligomycin. Imaging ATP depleted cells, we show that the disassembly of CHMP4b and ALIX observed in step 2 is ATP dependent. ATP depletion resulted in the recruitment of approximately 2-fold as many subunits of all ESCRTs. Resuming ATP production in cells, resulted in disassembly of the full ESCRT machinery which had been locked in place during ATP depletion. With some caveats, our experiments provide insight into the formation of the ESCRT machinery at the budding site of HIV during budding.
内体分选复合物需要运输蛋白（ESCRT）在细胞质远离芽的膜的泡中发挥作用。在这里，我们使用全内反射荧光（TIRF）显微镜来专门观察 ESCRTs（Alix、CHMP4b 和 VPS4）在 HIV Gag 病毒样颗粒（VLPs）出芽过程中的募集情况。我们以 200 毫秒的时间分辨率对出芽 VLPs 进行了 300 帧的成像。我们的数据显示了 ESCRT 动力学的三个阶段：1）募集，其中 Alix、CHMP4b 和 VPS4 的亚基以恒定的比例在 HIV Gag 病毒样颗粒的出芽部位募集，持续近 10 秒，然后 2）Alix 和 CHMP4b 的解体，而 VPS4 信号持续近 20 秒，然后 3）VPS4 的解体。我们假设在步骤 2 中观察到的解体是由 VPS4 催化的，由 ATP 水解提供动力。为了验证这一假设，我们使用（-）葡萄糖培养基、葡萄糖和寡霉素进行了 ATP 耗竭。对 ATP 耗竭细胞进行成像，我们表明步骤 2 中观察到的 CHMP4b 和 Alix 的解体是依赖于 ATP 的。ATP 耗竭导致所有 ESCRTs 的亚基募集增加约 2 倍。在细胞中恢复 ATP 的产生，导致在 ATP 耗竭期间锁定在原位的完整 ESCRT 机制解体。有一些限制，但我们的实验为了解 HIV 在出芽过程中 ESCRT 机制在出芽部位的形成提供了一些线索。
