Nanoscale dynamics and localization of single endogenous mRNAs in stress granules
Nanoscale dynamics and localization of single endogenous mRNAs in stress granules
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DOI：
10.1093/nar/gkae588
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发表时间：
2024-07-29
影响因子：
14.9
通讯作者：
Okabe，Kohki
中科院分区：
文献类型：
--
作者：
Sugawara，Ko;Uno，Shin-nosuke;Okabe，Kohki
应激颗粒（Stress granules，SGs）是哺乳动物细胞在应激状态下瞬时形成的胞质信使核糖核蛋白颗粒。虽然SG组件已被很好地表征，详细的见解内SGs的分子行为仍然没有得到解决。我们研究了纳米级的动力学和本地化的内源性mRNA结合单个mRNA跟踪和超分辨率本地化显微镜在SGs。首先，我们开发了一种跟踪SGs内单个mRNA的方法，揭示了尽管SGs中的mRNA主要是固定的（约40%），但它们也以受限的（约25%）或自由扩散的（约35%）方式移动。其次，超分辨定位显微镜显示，SGs中的mRNA分布不均匀，部分形成高密度簇。第三，我们同时进行了单个mRNA跟踪和超分辨率显微镜在SGs，表明单个mRNA的轨迹主要是发现周围的高密度集群。最后，使用实时超分辨率成像和单分子跟踪对应力消除过程中的mRNA定位和动力学进行定量分析。这些结果表明，SGs具有高度组织化的结构，能够在纳米级动态调节mRNA，这是负责有序形成和各种各样的功能SGs。
Stress granules (SGs) are cytoplasmic messenger ribonucleoprotein granules transiently formed in stressed mammalian cells. Although SG components have been well characterized, detailed insights into the molecular behavior inside SGs remain unresolved. We investigated nanoscale dynamics and localization of endogenous mRNAs in SGs combining single mRNA tracking and super-resolution localization microscopy. First, we developed a methodology for tracking single mRNAs within SGs, revealing that although mRNAs in SGs are mainly stationary (∼40%), they also move in a confined (∼25%) or freely diffusing (∼35%) manner. Second, the super-resolution localization microscopy showed that the mRNAs in SGs are heterogeneously distributed and partially form high-density clusters. Third, we simultaneously performed single mRNA tracking and super-resolution microscopy in SGs, demonstrating that single mRNA trajectories are mainly found around high-density clusters. Finally, a quantitative analysis of mRNA localization and dynamics during stress removal was conducted using live super-resolution imaging and single-molecule tracking. These results suggest that SGs have a highly organized structure that enables dynamic regulation of the mRNAs at the nanoscale, which is responsible for the ordered formation and the wide variety of functions of SGs.