Inaba Hironori, Imasaki Tsuyoshi, Aoyama Kazuhiro, Yoshihara Shogo, Takazaki Hiroko, Kato Takayuki, Goto Hidemasa, Mitsuoka Kaoru, Nitta Ryo, Nakata Takao
Department of Cell Biology, Graduate School of Medical and Dental Sciences, Institute of Science Tokyo, Tokyo 113-8510, Japan.
The Center for Brain Integration Research (CBIR), Institute of Science Tokyo, Tokyo 113-8510, Japan.
iScience. 2025 Apr 24;28(6):112529. doi: 10.1016/j.isci.2025.112529. eCollection 2025 Jun 20.
Lamellipodia are sheet-like protrusions essential for cell migration and endocytosis, but their ultrastructural dynamics remain poorly understood because conventional electron microscopy lacks temporal resolution. Here, we combined optogenetics with cryo-electron tomography (cryo-ET) to visualize the actin cytoskeleton and membrane structures during lamellipodia formation with temporal precision. Using photoactivatable-Rac1 (PA-Rac1) in COS-7 cells, we induced lamellipodia formation with a 2-min blue light irradiation, rapidly vitrified samples, and analyzed their ultrastructure with cryo-ET. We obtained 16 tomograms of lamellipodia at different degrees of extension from three cells. These revealed small protrusions with unbundled actin filaments, "mini filopodia" composed of short, bundled actin filaments at the leading edge, and actin bundles running nearly parallel to the leading edge within inner regions of lamellipodia, suggesting dynamic reorganizations of the actin cytoskeleton. This approach provides a powerful framework for elucidating the ultrastructural dynamics of cellular processes with precise temporal control.
片足是细胞迁移和内吞作用所必需的片状突起，但由于传统电子显微镜缺乏时间分辨率，其超微结构动力学仍知之甚少。在这里，我们将光遗传学与冷冻电子断层扫描（cryo-ET）相结合，以在时间精度上可视化片足形成过程中的肌动蛋白细胞骨架和膜结构。在COS-7细胞中使用光激活Rac1（PA-Rac1），我们通过2分钟的蓝光照射诱导片足形成，快速冷冻样品，并使用cryo-ET分析其超微结构。我们从三个细胞中获得了不同伸展程度的片足的16张断层扫描图。这些图揭示了具有未束状肌动蛋白丝的小突起、前缘由短的束状肌动蛋白丝组成的“微型丝状伪足”，以及在片足内部区域几乎平行于前缘延伸的肌动蛋白束，这表明肌动蛋白细胞骨架发生了动态重组。这种方法为在精确的时间控制下阐明细胞过程的超微结构动力学提供了一个强大的框架。
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