Schoenenberger C-A, Buchmeier S, Boerries M, Sütterlin R, Aebi U, Jockusch B M
M.E. Müller Institute for Structural Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
J Struct Biol. 2005 Dec;152(3):157-68. doi: 10.1016/j.jsb.2005.09.003. Epub 2005 Oct 21.
For many years the existence of actin in the nucleus has been doubted because of the lack of phalloidin staining as well as the failure to document nuclear actin filaments by electron microscopy. More recent findings reveal actin to be a component of chromatin remodeling complexes and of the machinery involved in RNA synthesis and transport. With distinct functions for nuclear actin emerging, the quest for its conformation and oligomeric/polymeric structure in the nucleus has resumed importance. We used chemically cross-linked 'lower dimer' (LD) to generate mouse monoclonal antibodies specific for different actin conformations. One of the resulting antibodies, termed 1C7, recognizes an epitope that is buried in the F-actin filament, but is surface-exposed in G-actin as well as in the LD. In immunofluorescence studies with different cell lines, 1C7 selectively reacts with non-filamentous actin in the cytoplasm. In addition, it detects a discrete form of actin in the nucleus, which is different from the nuclear actin revealed by the previously described 2G2 [Gonsior, S.M., Platz, S., Buchmeier, S., Scheer, U., Jockusch, B.M., Hinssen, H., 1999. J. Cell Sci. 112, 797]. Upon latrunculin-induced disassembly of the filamentous cytoskeleton in Rat2 fibroblasts, we observed a perinuclear accumulation of the 1C7-reactive actin conformation. In addition, latrunculin treatment led to the assembly of phalloidin-staining actin structures in chromatin-free regions of the nucleus in these cells. Our results indicate that distinct actin conformations and/or structures are present in the nucleus and the cytoplasm of different cell types and that their distribution varies in response to external signals.
多年来，由于缺乏鬼笔环肽染色以及未能通过电子显微镜记录核肌动蛋白丝，细胞核中肌动蛋白的存在一直受到质疑。最近的研究结果表明，肌动蛋白是染色质重塑复合物以及参与RNA合成和运输的机制的组成部分。随着核肌动蛋白独特功能的出现，对其在细胞核中的构象和寡聚/聚合结构的探索再次变得重要起来。我们使用化学交联的“低聚物二聚体”（LD）来生成针对不同肌动蛋白构象的小鼠单克隆抗体。其中一种产生的抗体，称为1C7，识别一个埋藏在F-肌动蛋白丝中的表位，但在G-肌动蛋白以及LD中则暴露于表面。在对不同细胞系的免疫荧光研究中，1C7与细胞质中的非丝状肌动蛋白选择性反应。此外，它在细胞核中检测到一种离散形式的肌动蛋白，这与先前描述的2G2所揭示的核肌动蛋白不同[Gonsior, S.M., Platz, S., Buchmeier, S., Scheer, U., Jockusch, B.M., Hinssen, H., 1999. J. Cell Sci. 112, 797]。在用拉特环菌素诱导大鼠2成纤维细胞中丝状细胞骨架解体后，我们观察到1C7反应性肌动蛋白构象在核周积累。此外，拉特环菌素处理导致这些细胞细胞核无染色质区域中鬼笔环肽染色的肌动蛋白结构组装。我们的结果表明，不同细胞类型的细胞核和细胞质中存在不同的肌动蛋白构象和/或结构，并且它们的分布会因外部信号而变化。
