Visualization of endogenous nuclear F-actin in mouse embryos reveals abnormal actin assembly after somatic cell nuclear transfer.
Visualization of endogenous nuclear F-actin in mouse embryos reveals abnormal actin assembly after somatic cell nuclear transfer.
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DOI：
10.1093/jb/mvaa125
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发表时间：
2020-11
影响因子：
2.7
通讯作者：
T. Shindo;Shunya Ihashi;Yuko Sakamoto;Tomomi Okuno;Junko Tomikawa;Kei Miyamoto
中科院分区：
文献类型：
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作者：
T. Shindo;Shunya Ihashi;Yuko Sakamoto;Tomomi Okuno;Junko Tomikawa;Kei Miyamoto
细胞核中的肌动蛋白，简称核肌动蛋白，参与各种核事件。核肌动蛋白以球状（G-肌动蛋白）和丝状（F-肌动蛋白）的形式存在，核肌动蛋白的动态组装/拆卸深刻地影响核功能。然而，它仍然是具有挑战性的观察内源性核F-肌动蛋白。在这里，我们提出了一个条件，可视化内源性核F-肌动蛋白的小鼠受精卵使用不同的固定方法。用多聚甲醛固定并用荧光结合的鬼笔环肽处理的合子在其原核中显示出短和长的肌动蛋白丝。短的核肌动蛋白丝是鬼笔环肽染色的特征，而不是通过固定过程切断肌动蛋白丝的结果，因为用核肌动蛋白染色体探测的长的核肌动蛋白丝在用多聚甲醛固定后不会分解成短的丝。此外，我们发现，核肌动蛋白组装体细胞核移植（SCNT）后受损，表明异常核骨架结构的SCNT胚胎。综上所述，我们提出的用鬼笔环肽显示核F-肌动蛋白的方法可以用来观察核肌动蛋白的组装状态，并揭示克隆小鼠胚胎中肌动蛋白核骨架结构的不正确重编程。
Actin in the nucleus, referred to as nuclear actin, is involved in a variety of nuclear events. Nuclear actin is present as a globular (G-actin) and filamentous form (F-actin), and dynamic assembly/disassembly of nuclear actin profoundly affects nuclear functions. However, it is still challenging to observe endogenous nuclear F-actin. Here, we present a condition to visualize endogenous nuclear F-actin of mouse zygotes using different fixation methods. Zygotes fixed with paraformaldehyde and treated with fluorescently conjugated phalloidin show both short and long actin filaments in their pronuclei. Short nuclear actin filaments are characteristic of phalloidin staining, rather than the consequence of severing actin filaments by the fixation process, since long nuclear actin filaments probed with the nuclear actin chromobody are not disassembled into short filaments after fixation with paraformaldehyde. Furthermore, we find that nuclear actin assembly is impaired after somatic cell nuclear transfer (SCNT), suggesting abnormal nucleoskeleton structures in SCNT embryos. Taken together, our presented method for visualizing nuclear F-actin with phalloidin can be used to observe the states of nuclear actin assembly, and revealed improper reprogramming of actin nucleoskeleton structures in cloned mouse embryos.