Key Laboratory of Horticultural Plant Biology (MOE), College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, Hubei Province, China; Interdisciplinary Sciences Research Institute, Huazhong Agricultural University, Wuhan 430007, China; Department of Biosciences, Durham University, South Road, Durham DH1 3LE, UK.
Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, 06120 Halle, Germany.
Curr Biol. 2021 Mar 22;31(6):1251-1260.e4. doi: 10.1016/j.cub.2020.12.009. Epub 2021 Feb 15.
In plants, the cortical endoplasmic reticulum (ER) network is connected to the plasma membrane (PM) through the ER-PM contact sites (EPCSs), whose structures are maintained by EPCS resident proteins and the cytoskeleton. Strong co-alignment between EPCSs and the cytoskeleton is observed in plants, but little is known of how the cytoskeleton is maintained and regulated at the EPCS. Here, we have used a yeast-two-hybrid screen and subsequent in vivo interaction studies in plants by fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) analysis to identify two microtubule binding proteins, KLCR1 (kinesin-light-chain-related protein 1) and IQD2 (IQ67-domain 2), that interact with the actin binding protein NET3C and form a component of plant EPCS that mediates the link between the actin and microtubule networks. The NET3C-KLCR1-IQD2 module, acting as an actin-microtubule bridging complex, has a direct influence on ER morphology and EPCS structure. Their loss-of-function mutants, net3a/NET3C RNAi, klcr1, or iqd2, exhibit defects in pavement cell morphology, which we suggest is linked to the disorganization of both actin filaments and microtubules. In conclusion, our results reveal a novel cytoskeletal-associated complex, which is essential for the maintenance and organization of cytoskeletal structure and ER morphology at the EPCS and for normal plant cell morphogenesis.
在植物中，皮质内质网（ER）网络通过 ER-PM 接触位点（EPCS）与质膜（PM）相连，其结构由 EPCS 驻留蛋白和细胞骨架维持。在植物中观察到 EPCS 与细胞骨架之间的强共定位，但对于细胞骨架如何在 EPCS 处得到维持和调控知之甚少。在这里，我们使用酵母双杂交筛选和随后通过荧光共振能量转移（FRET）-荧光寿命成像显微镜（FLIM）分析在植物中的体内相互作用研究，鉴定了两个微管结合蛋白，KLCR1（驱动蛋白轻链相关蛋白 1）和 IQD2（IQ67 结构域 2），它们与肌动蛋白结合蛋白 NET3C 相互作用并形成植物 EPCS 的一个组成部分，介导了肌动蛋白和微管网络之间的联系。NET3C-KLCR1-IQD2 模块作为肌动蛋白-微管桥接复合物，对 ER 形态和 EPCS 结构有直接影响。它们的功能丧失突变体 net3a/NET3C RNAi、klcr1 或 iqd2 表现出 pavement 细胞形态的缺陷，我们认为这与肌动蛋白丝和微管的紊乱有关。总之，我们的结果揭示了一种新的细胞骨架相关复合物，对于 EPCS 处细胞骨架结构和 ER 形态的维持和组织以及正常的植物细胞形态发生是必不可少的。
