The endoplasmic reticulum (ER) membrane-resident stress sensor inositol-requiring enzyme 1 (IRE1) governs the most evolutionarily conserved branch of the unfolded protein response. Upon sensing an accumulation of unfolded proteins in the ER lumen, IRE1 activates its cytoplasmic kinase and ribonuclease domains to transduce the signal. IRE1 activity correlates with its assembly into large clusters, yet the biophysical characteristics of IRE1 clusters remain poorly characterized. We combined superresolution microscopy, single-particle tracking, fluorescence recovery, and photoconversion to examine IRE1 clustering quantitatively in living human and mouse cells. Our results revealed that: 1) In contrast to qualitative impressions gleaned from microscopic images, IRE1 clusters comprise only a small fraction (∼5%) of the total IRE1 in the cell; 2) IRE1 clusters have complex topologies that display features of higher-order organization; 3) IRE1 clusters contain a diffusionally constrained core, indicating that they are not phase-separated liquid condensates; 4) IRE1 molecules in clusters remain diffusionally accessible to the free pool of IRE1 molecules in the general ER network; 5) when IRE1 clusters disappear at later time points of ER stress as IRE1 signaling attenuates, their constituent molecules are released back into the ER network and not degraded; 6) IRE1 cluster assembly and disassembly are mechanistically distinct; and 7) IRE1 clusters' mobility is nearly independent of cluster size. Taken together, these insights define the clusters as dynamic assemblies with unique properties. The analysis tools developed for this study will be widely applicable to investigations of clustering behaviors in other signaling proteins.

中文翻译：

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定量显微镜揭示了簇状IRE1α的动力学和命运。

内质网（ER）膜驻留应力传感器肌醇需要酶1（IRE1）控制未折叠的蛋白反应中最进化保守的分支。感应到ER内腔中未折叠蛋白的积累后，IRE1激活其胞质激酶和核糖核酸酶结构域以转导信号。IRE1活性与其组装成大簇有关，但是IRE1簇的生物物理特征仍然很差。我们结合了超分辨率显微镜，单粒子跟踪，荧光恢复和光转换技术，以定量检测活人和小鼠细胞中的IRE1簇。我们的结果表明：1）与从显微图像中获得的定性印象相反，IRE1簇仅占细胞中全部IRE1的一小部分（约5％）；2）IRE1集群具有复杂的拓扑结构，这些拓扑结构显示出高阶组织的功能；3）IRE1团簇包含一个扩散约束核，表明它们不是相分离的液体冷凝物；4）簇中的IRE1分子仍然可以扩散到一般ER网络中的IRE1分子的自由池中；5）当IRE1簇随着IRE1信号减弱而在ER应力的较晚时间点消失时，其组成分子被释放回ER网络中而不降解。6）IRE1群集的组装和拆卸在机械上是不同的；7）IRE1群集的移动性几乎与群集大小无关。综上所述，这些见解将群集定义为具有独特属性的动态程序集。