One third of all human proteins are either transmembrane or soluble secretory proteins that first target the endoplasmic reticulum (ER). These proteins subsequently leave the ER and enter the Golgi apparatus via ER-Golgi intermediate vesicular structures. Live-cell imaging of cargos fused to fluorescent proteins (FPs) enables the high-resolution visualization and characterization of secretory transport processes. Here, we performed fluorescence time-lapse imaging to assess the Ca²⁺ and energy dependency of ER-to-Golgi transport in living HeLa cells, a cancer cell model which has been well investigated. Our data revealed that ER-to-Golgi transport remained highly efficient in the absence of ATP-generating substrates, despite clear reductions in cytosolic and mitochondrial ATP levels under these energy stress conditions. However, cell treatment with 2-deoxy-D-glucose (2-DG), which severely diminished subcellular ATP levels, abolished ER-to-Golgi transport. Interestingly, while 2-DG elevated cytosolic Ca²⁺ levels and reduced long-distance movements of glycosylphosphatidylinositol (GPI)-positive vesicles, robust short-term ER Ca²⁺ mobilizations, which strongly affected the motility of these vesicles, did not considerably impair ER-to-Golgi transport. In summary, we highlight that ER-to-Golgi transport in HeLa cells remains functional despite high energy and Ca²⁺ stress levels.

中文翻译：

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HeLa 细胞中内质网到高尔基体的运输对 Ca2+ 和能量应激表现出高弹性

三分之一的人类蛋白质是跨膜蛋白或可溶性分泌蛋白，它们首先靶向内质网 (ER)。这些蛋白质随后离开内质网并通过内质网-高尔基体中间囊泡结构进入高尔基体。与荧光蛋白 (FP) 融合的货物的活细胞成像能够实现分泌运输过程的高分辨率可视化和表征。在这里，我们进行了荧光延时成像，以评估活 HeLa 细胞（一种已得到充分研究的癌细胞模型）中内质网至高尔基体转运的Ca ^{2+和能量依赖性。}我们的数据显示，尽管在这些能量应激条件下细胞质和线粒体 ATP 水平明显降低，但在缺乏 ATP 生成底物的情况下，内质网到高尔基体的转运仍然保持高效。然而，用 2-脱氧-D-葡萄糖 (2-DG) 处理细胞会严重降低亚细胞 ATP 水平，从而消除了内质网到高尔基体的转运。有趣的是，虽然 2-DG 提高了胞浆 Ca ²⁺水平并减少了糖基磷脂酰肌醇 (GPI) 阳性囊泡的长距离运动，但强烈影响这些囊泡运动性的强大的短期 ER Ca ²⁺动员并没有显着损害ER 到高尔基体的运输。^{总之，我们强调，尽管能量和 Ca 2+}应激水平较高，但 HeLa 细胞中的 ER 至高尔基体转运仍保持功能。