Nuclear actin and protein 4.1:: Essential interactions during nuclear assembly in vitro
Nuclear actin and protein 4.1:: Essential interactions during nuclear assembly in vitro
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DOI：
10.1073/pnas.1934680100
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发表时间：
2003-09-16
影响因子：
11.1
通讯作者：
Heald, R
中科院分区：
文献类型：
--
作者：
Krauss, SW;Chen, C;Heald, R
结构蛋白4.1在人红细胞膜骨架的血影蛋白-肌动蛋白晶格内具有重要的相互作用，也广泛分布在有核细胞的不同细胞内位点。我们以前表明，4.1是必不可少的功能核在体外组装和能力的4.1结合肌动蛋白是必需的。在这里，我们报告说，4.1和肌动蛋白共定位在哺乳动物细胞核中使用荧光显微镜，并通过更高分辨率的洗涤剂提取的细胞整体安装电子显微镜，相关的核丝。我们还设计了一个无细胞的测定，使用非洲爪蟾卵提取物含有荧光肌动蛋白遵循肌动蛋白在核组装。通过在非扰动条件下直接成像肌动蛋白，总的核肌动蛋白群体被保留并相对于完整的染色质原位可视化。当染色质和核孔开始组装时，我们最初检测到肌动蛋白。当核纤层组装时，但在DNA合成之前，肌动蛋白以网状图案分布在整个核中。当肌动蛋白开始在细胞核中积累时，也检测到蛋白4.1表位，产生弥漫性一致模式。随着细胞核的成熟，肌动蛋白被检测到与4.1表位一致，也独立于4.1表位。为了测试核肌动蛋白的获取是否是核组装所必需的，在核组装期间将肌动蛋白抑制剂latrunculin A添加到爪蟾卵提取物中。Latrunculin A强烈扰动核组装，产生扭曲的核结构，既不含肌动蛋白也不含蛋白4.1。我们的研究结果表明，肌动蛋白以及4.1是必要的核组装和4.1-肌动蛋白的相互作用可能是至关重要的。
Structural protein 4.1, which has crucial interactions within the spectrin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher-resolution detergent-extracted cell whole-mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under nonperturbing conditions, the total nuclear actin population is retained and visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As nuclear lamina assembled, but preceding DNA synthesis, actin distributed in a reticulated pattern throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.