Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
Bioinformatics and Structural Biology Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037.
Proc Natl Acad Sci U S A. 2019 Jan 22;116(4):1267-1272. doi: 10.1073/pnas.1808830116. Epub 2019 Jan 10.
Rac1 activation is at the core of signaling pathways regulating polarized cell migration. So far, it has not been possible to directly explore the structural changes triggered by Rac1 activation at the molecular level. Here, through a multiscale imaging workflow that combines biosensor imaging of Rac1 dynamics with electron cryotomography, we identified, within the crowded environment of eukaryotic cells, a unique nanoscale architecture of a flexible, signal-dependent actin structure. In cell regions with high Rac1 activity, we found a structural regime that spans from the ventral membrane up to a height of ∼60 nm above that membrane, composed of directionally unaligned, densely packed actin filaments, most shorter than 150 nm. This unique Rac1-induced morphology is markedly different from the dendritic network architecture in which relatively short filaments emanate from existing, longer actin filaments. These Rac1-mediated scaffold assemblies are devoid of large macromolecules such as ribosomes or other filament types, which are abundant at the periphery and within the remainder of the imaged volumes. Cessation of Rac1 activity induces a complete and rapid structural transition, leading to the absence of detectable remnants of such structures within 150 s, providing direct structural evidence for rapid actin filament network turnover induced by GTPase signaling events. It is tempting to speculate that this highly dynamical nanoscaffold system is sensitive to local spatial cues, thus serving to support the formation of more complex actin filament architectures-such as those mandated by epithelial-mesenchymal transition, for example-or resetting the region by completely dissipating.
Rac1 的激活是调节极化细胞迁移的信号通路的核心。到目前为止，还不可能直接在分子水平上探索 Rac1 激活引发的结构变化。在这里，我们通过一种多尺度成像工作流程，将 Rac1 动力学的生物传感器成像与电子晶体断层扫描相结合，在真核细胞拥挤的环境中，确定了一种独特的、灵活的、信号依赖性的肌动蛋白结构的纳米尺度结构。在 Rac1 活性较高的细胞区域，我们发现了一种结构状态，从腹膜延伸到该膜上方约 60nm 的高度，由定向未对齐、密集排列的肌动蛋白丝组成，大多数长度短于 150nm。这种独特的 Rac1 诱导形态与从现有较长肌动蛋白丝发出的相对较短的丝状结构组成的树突状网络架构明显不同。这些 Rac1 介导的支架组装不包含核糖体或其他类型的细丝等大量大分子，这些大分子在边缘和成像体积的其余部分很丰富。Rac1 活性的停止会诱导完全和快速的结构转变，导致在 150s 内这些结构没有可检测到的残留物，这为 GTPase 信号事件诱导的肌动蛋白丝网络快速周转提供了直接的结构证据。人们不禁推测，这种高度动态的纳米支架系统对局部空间线索敏感，从而支持形成更复杂的肌动蛋白丝结构，例如上皮-间充质转化所需要的结构，或者通过完全耗散来重置该区域。
