Cryo-ET of actin cytoskeleton and membrane structure in lamellipodia formation using optogenetics

IF 4.6 2区综合性期刊 Q1 MULTIDISCIPLINARY SCIENCES

iScience Pub Date : 2025-04-24 DOI:10.1016/j.isci.2025.112529

Hironori Inaba , Tsuyoshi Imasaki , Kazuhiro Aoyama , Shogo Yoshihara , Hiroko Takazaki , Takayuki Kato , Hidemasa Goto , Kaoru Mitsuoka , Ryo Nitta , Takao Nakata

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引用次数: 0

Abstract

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.

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利用光遗传学技术研究板足形成过程中肌动蛋白细胞骨架和膜结构

板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，对其超微结构动力学仍然知之甚少。在这里，我们将光遗传学与冷冻电子断层扫描（cryo-ET）相结合，以时间精度观察板足形成过程中的肌动蛋白细胞骨架和膜结构。我们利用COS-7细胞中的光活化因子rac1 (PA-Rac1)，在2分钟蓝光照射下诱导板状足形成，快速玻璃化样品，并用冷冻et分析其超微结构。我们从3个细胞中获得了16张不同伸展程度的板足断层图。这些图像显示了肌动蛋白纤维未捆绑的小突起，由前缘短而捆绑的肌动蛋白丝组成的“迷你丝状足”，以及肌动蛋白束在板足内部区域内几乎平行于前缘，表明肌动蛋白细胞骨架的动态重组。这种方法为阐明具有精确时间控制的细胞过程的超微结构动力学提供了一个强有力的框架。

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来源期刊

iScience Multidisciplinary-Multidisciplinary

CiteScore

7.20

自引率

1.70%

发文量

1972

审稿时长

6 weeks

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