Zhang Yanshu, Yoshida Aiko, Sakai Nobuaki, Uekusa Yoshitsugu, Kumeta Masahiro, Yoshimura Shige H
Graduate School of Biostudies, Kyoto University, Yoshida-konoe, Sakyo-ku, Kyoto 606-8501, Japan.
R&D Group, Olympus Corp., Tokyo 192-8512, Japan.
Microscopy (Oxf). 2017 Aug 1;66(4):272-282. doi: 10.1093/jmicro/dfx015.
Together with lamellipodia and stress fibers, a dynamic network of actin filaments in the cell cortex plays a major role in the maintenance of cell morphology and motility. In contrast to lamellipodia, which have been well studied in various motile cells, the dynamics of actin filaments in the cell cortex have not yet been clarified due to a lack of proper imaging techniques. Here, we utilized high-speed atomic force microscopy for live-cell imaging and analyzed cortical actin dynamics in living cells. We successfully measured the polymerization rate and the frequency of filament synthesis in living COS-7 cells, and examined the associated effects of various inhibitors and actin-binding proteins. Actin filaments are synthesized beneath the plasma membrane and eventually descend into the cytoplasm. The inhibitors, cytochalasin B inhibited the polymerization, while jasplakinolide, inhibited the turnover of actin filaments as well as descension of the newly synthesized filaments, suggesting that actin polymerization near the membrane drives turnover of the cortical actin meshwork. We also determined how actin turnover is maintained and regulated by the free G-actin pool and G-actin binding proteins such as profilin and thymosin β4, and found that only a small amount of free G-actin was present in the cortex. Finally, we analyzed several different cell types, and found that the mesh size and the orientation of actin filaments were highly divergent, indicating the involvement of various actin-binding proteins in the maintenance and regulation of cortical actin architecture in each cell type.
细胞皮层中由肌动蛋白丝构成的动态网络与片状伪足和应力纤维一起，在维持细胞形态和运动性方面发挥着重要作用。与在各种运动细胞中得到充分研究的片状伪足不同，由于缺乏合适的成像技术，细胞皮层中肌动蛋白丝的动力学尚未得到阐明。在这里，我们利用高速原子力显微镜对活细胞进行成像，并分析了活细胞中皮层肌动蛋白的动力学。我们成功测量了活的COS - 7细胞中肌动蛋白丝的聚合速率和丝合成频率，并研究了各种抑制剂和肌动蛋白结合蛋白的相关作用。肌动蛋白丝在质膜下方合成，最终下降到细胞质中。抑制剂细胞松弛素B抑制聚合作用，而茉莉酸内酯则抑制肌动蛋白丝的周转以及新合成丝的下降，这表明膜附近的肌动蛋白聚合驱动皮层肌动蛋白网络的周转。我们还确定了游离G - 肌动蛋白池以及诸如丝切蛋白和胸腺素β4等G - 肌动蛋白结合蛋白如何维持和调节肌动蛋白周转，并且发现皮层中仅存在少量游离G - 肌动蛋白。最后，我们分析了几种不同的细胞类型，发现肌动蛋白丝的网孔大小和方向差异很大，这表明各种肌动蛋白结合蛋白参与了每种细胞类型中皮层肌动蛋白结构的维持和调节。
