Olson Christopher, Zhang Pengyang, Ku Joy, Flojo Renceh, Boyes Darin, Lu Biao
Department of Bioengineering, Santa Clara University, 500 El Camino Real, Santa Clara, CA, 95053, USA.
Department of Biology, Santa Clara University, 500 El Camino Real, Santa Clara, CA, 95053, USA.
Biol Proced Online. 2023 Sep 20;25(1):25. doi: 10.1186/s12575-023-00219-w.
Exosomes, a special subtype of extracellular vesicles derived from human cells, serve as vital mediators of intercellular communication by transporting diverse bioactive cargos, including proteins and enzymes. However, the underlying mechanisms governing exosome secretion and regulation remain poorly understood. In this study, we employed a dual-reporter system consisting of bioluminescent Gaussia luciferase and fluorescent proteins to investigate the dynamics and regulation of exosome secretion in cultured human cells.
Our results demonstrated that the engineered dual-reporters effectively monitored both exosome-mediated and ER-Golgi-mediated secretory pathways in a specific and quantitative manner. Notably, we observed distinct characteristics of exosome-mediated protein secretion, including significantly lower capacity and different dynamics compared to the ER-Golgi pathway. This phenomenon was observed in human kidney 293T cells and liver HepG2 cells, emphasizing the conserved nature of exosome-mediated secretion across cell types. Furthermore, we investigated the impact of brefeldin A (BFA), an inhibitor of ER-to-Golgi membrane trafficking, on protein secretion. Interestingly, BFA inhibited protein secretion via the ER-Golgi pathway while stimulating exosome-mediated protein secretion under same experimental conditions.
Collectively, our study highlights the utility of the dual-reporter system for real-time monitoring and quantitative analysis of protein secretion through conventional ER-Golgi and unconventional exosome pathways. Moreover, our findings unveil distinct features of exosome-mediated protein secretion, shedding light on its differential capacity, dynamics, and regulatory mechanisms compared to ER-Golgi-mediated proteins in human cells.
外泌体是源自人类细胞的细胞外囊泡的一种特殊亚型，通过运输包括蛋白质和酶在内的多种生物活性物质，充当细胞间通讯的重要介质。然而，外泌体分泌和调控的潜在机制仍知之甚少。在本研究中，我们采用了一种由生物发光的高斯荧光素酶和荧光蛋白组成的双报告系统，来研究培养的人类细胞中外泌体分泌的动力学和调控。
我们的结果表明，工程化的双报告基因能够以特异性和定量的方式有效监测外泌体介导和内质网-高尔基体介导的分泌途径。值得注意的是，我们观察到外泌体介导的蛋白质分泌具有独特的特征，包括与内质网-高尔基体途径相比，其分泌能力显著降低且动力学不同。在人肾293T细胞和肝HepG2细胞中均观察到这种现象，强调了外泌体介导的分泌在不同细胞类型中的保守性质。此外，我们研究了内质网到高尔基体膜转运抑制剂布雷菲德菌素A（BFA）对蛋白质分泌的影响。有趣的是，在相同实验条件下，BFA抑制了通过内质网-高尔基体途径的蛋白质分泌，同时刺激了外泌体介导的蛋白质分泌。
总体而言，我们的研究突出了双报告系统在通过传统内质网-高尔基体和非传统外泌体途径实时监测和定量分析蛋白质分泌方面的实用性。此外，我们的发现揭示了外泌体介导的蛋白质分泌的独特特征，阐明了其与人细胞中内质网-高尔基体介导的蛋白质相比在分泌能力、动力学和调控机制上的差异。
