Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Mol Biol Cell. 2010 Jun 15;21(12):1909-21. doi: 10.1091/mbc.e09-12-1066. Epub 2010 Apr 21.
Accumulation of misfolded secretory proteins causes cellular stress and induces the endoplasmic reticulum (ER) stress pathway, the unfolded protein response (UPR). Although the UPR has been extensively studied, little is known about the molecular changes that distinguish the homeostatic and stressed ER. The increase in levels of misfolded proteins and formation of complexes with chaperones during ER stress are predicted to further crowd the already crowded ER lumen. Surprisingly, using live cell fluorescence microscopy and an inert ER reporter, we find the crowdedness of stressed ER, treated acutely with tunicamycin or DTT, either is comparable to homeostasis or significantly decreases in multiple cell types. In contrast, photobleaching experiments revealed a GFP-tagged variant of the ER chaperone BiP rapidly undergoes a reversible quantitative decrease in diffusion as misfolded proteins accumulate. BiP mobility is sensitive to exceptionally low levels of misfolded protein stressors and can detect intermediate states of BiP availability. Decreased BiP availability temporally correlates with UPR markers, but restoration of BiP availability correlates less well. Thus, BiP availability represents a novel and powerful tool for reporting global secretory protein misfolding levels and investigating the molecular events of ER stress in single cells, independent of traditional UPR markers.
错误折叠的分泌蛋白的积累会导致细胞应激，并诱导内质网 (ER) 应激途径，即未折叠蛋白反应 (UPR)。尽管 UPR 已经被广泛研究，但对于区分内质网稳态和应激状态的分子变化知之甚少。内质网应激期间错误折叠蛋白水平的增加和与伴侣蛋白形成复合物，预计会进一步使已经拥挤的内质网腔更加拥挤。令人惊讶的是，使用活细胞荧光显微镜和惰性内质网报告器，我们发现用衣霉素或 DTT 急性处理的应激内质网的拥挤程度与稳态相当，或者在多种细胞类型中显著降低。相比之下，光漂白实验表明，GFP 标记的内质网伴侣蛋白 BiP 的变体在错误折叠蛋白积累时会迅速经历扩散的可逆定量减少。BiP 的流动性对异常低水平的错误折叠蛋白应激原敏感，并可以检测到 BiP 可用性的中间状态。BiP 可用性的降低与 UPR 标志物时间上相关，但 BiP 可用性的恢复相关性较差。因此，BiP 可用性是一种新的强大工具，可用于报告全局分泌蛋白错误折叠水平，并在单细胞中研究内质网应激的分子事件，而不依赖于传统的 UPR 标志物。
