Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Ibaraki, 305-8565, Japan.
Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, 305-8572, Japan.
Sci Rep. 2018 Mar 12;8(1):4381. doi: 10.1038/s41598-018-22707-w.
Flowering plants express multiple actin isoforms. Previous studies suggest that individual actin isoforms have specific functions; however, the subcellular localization of actin isoforms in plant cells remains obscure. Here, we transiently expressed and observed major Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, as fluorescent-fusion proteins. By optimizing the linker sequence between fluorescent protein and actin, we succeeded in observing filaments that contained these expressed actin isoforms fused with green fluorescent protein (GFP) in Arabidopsis protoplasts. Different colored fluorescent proteins fused with AtACT2 and AtACT7 and co-expressed in Nicotiana benthamiana mesophyll cells co-polymerized in a segregated manner along filaments. In epidermal cells, surprisingly, AtACT2 and AtACT7 tended to polymerize into different types of filaments. AtACT2 was incorporated into thinner filaments, whereas AtACT7 was incorporated into thick bundles. We conclude that different actin isoforms are capable of constructing unique filament arrays, depending on the cell type or tissue. Interestingly, staining patterns induced by two indirect actin filament probes, Lifeact and mTalin1, were different between filaments containing AtACT2 and those containing AtACT7. We suggest that filaments containing different actin isoforms bind specific actin-binding proteins in vivo, since the two probes comprise actin-binding domains from different actin-binding proteins.
植物开花表达多种肌动蛋白同工型。先前的研究表明，个别肌动蛋白同工型具有特定的功能；然而，植物细胞中肌动蛋白同工型的亚细胞定位仍然不清楚。在这里，我们通过瞬时表达和观察将主要的拟南芥营养肌动蛋白同工型 AtACT2 和 AtACT7 作为荧光融合蛋白来研究。通过优化荧光蛋白和肌动蛋白之间的连接序列，我们成功地观察到了含有这些表达的肌动蛋白同工型与绿色荧光蛋白 (GFP) 融合的纤维在拟南芥原生质体中。不同颜色的荧光蛋白与 AtACT2 和 AtACT7 融合并在本氏烟叶肉细胞中共表达，沿纤维以分隔的方式共聚合。令人惊讶的是，在表皮细胞中，AtACT2 和 AtACT7 倾向于聚合形成不同类型的纤维。AtACT2 被掺入到更细的纤维中，而 AtACT7 则被掺入到厚束中。我们得出的结论是，不同的肌动蛋白同工型能够根据细胞类型或组织构建独特的纤维阵列。有趣的是，Lifeact 和 mTalin1 这两种间接肌动蛋白纤维探针诱导的染色模式在含有 AtACT2 的纤维和含有 AtACT7 的纤维之间存在差异。我们认为，含有不同肌动蛋白同工型的纤维在体内结合特定的肌动蛋白结合蛋白，因为这两种探针包含来自不同肌动蛋白结合蛋白的肌动蛋白结合结构域。
