Holzinger Andreas, Blaas Kathrin
Functional Plant Biology, Institute of Botany, University of Innsbruck, Sternwartestraße 15, 6020, Innsbruck, Austria.
Methods Mol Biol. 2016;1365:243-61. doi: 10.1007/978-1-4939-3124-8_13.
This chapter gives an overview of the most common F-actin-perturbing substances that are used to study actin dynamics in living plant cells in studies on morphogenesis, motility, organelle movement, or when apoptosis has to be induced. These substances can be divided into two major subclasses: F-actin-stabilizing and -polymerizing substances like jasplakinolide and chondramides and F-actin-severing compounds like chytochalasins and latrunculins. Jasplakinolide was originally isolated form a marine sponge, and can now be synthesized and has become commercially available, which is responsible for its wide distribution as membrane-permeable F-actin-stabilizing and -polymerizing agent, which may even have anticancer activities. Cytochalasins, derived from fungi, show an F-actin-severing function and many derivatives are commercially available (A, B, C, D, E, H, J), also making it a widely used compound for F-actin disruption. The same can be stated for latrunculins (A, B), derived from red sea sponges; however the mode of action is different by binding to G-actin and inhibiting incorporation into the filament. In the case of swinholide a stable complex with actin dimers is formed resulting also in severing of F-actin. For influencing F-actin dynamics in plant cells only membrane permeable drugs are useful in a broad range. We however introduce also the phallotoxins and synthetic derivatives, as they are widely used to visualize F-actin in fixed cells. A particular uptake mechanism has been shown for hepatocytes, but has also been described in siphonal giant algae. In the present chapter the focus is set on F-actin dynamics in plant cells where alterations in cytoplasmic streaming can be particularly well studied; however methods by fluorescence applications including phalloidin and antibody staining as well as immunofluorescence-localization of the inhibitor drugs are given.
本章概述了最常见的F-肌动蛋白干扰物质,这些物质用于在形态发生、运动、细胞器运动研究中或必须诱导细胞凋亡时,研究活植物细胞中的肌动蛋白动力学。这些物质可分为两大类:F-肌动蛋白稳定和聚合物质,如茉莉素内酯和软骨酰胺;以及F-肌动蛋白切断化合物,如细胞松弛素和拉春库林。茉莉素内酯最初是从一种海洋海绵中分离出来的,现在可以合成并已商业化,这使得它作为一种膜渗透性F-肌动蛋白稳定和聚合剂广泛分布,甚至可能具有抗癌活性。细胞松弛素来源于真菌,具有F-肌动蛋白切断功能,许多衍生物都有商业供应(A、B、C、D、E、H、J),这也使其成为一种广泛用于破坏F-肌动蛋白的化合物。拉春库林(A、B)也是如此,它来源于红海海绵;然而,其作用方式不同,它通过与G-肌动蛋白结合并抑制其掺入细丝中。在swinholide的情况下,会与肌动蛋白二聚体形成稳定的复合物,也会导致F-肌动蛋白的切断。对于影响植物细胞中的F-肌动蛋白动力学,只有膜渗透性药物在广泛范围内是有用的。然而,我们也介绍了鬼笔毒素和合成衍生物,因为它们广泛用于在固定细胞中可视化F-肌动蛋白。肝细胞已显示出一种特殊的摄取机制,在管形巨型藻类中也有描述。在本章中,重点是植物细胞中的F-肌动蛋白动力学,在那里可以特别好地研究细胞质流动的变化;然而,也给出了包括鬼笔环肽和抗体染色在内的荧光应用方法以及抑制剂药物的免疫荧光定位方法。