Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells
Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells
复制标题
单分子荧光成像可量化活植物细胞中的膜蛋白动力学和寡聚化
DOI：
10.1038/nprot.2015.132
复制
发表时间：
2015-12-01
期刊：
影响因子：
14.8
通讯作者：
Lin, Jinxing
中科院分区：
文献类型：
--
作者：
Wang, Xiaohua;Li, Xiaojuan;Lin, Jinxing
测量蛋白质的迁移率和相互作用是理解细胞信号机制的关键；然而，定量分析活的植物细胞中的蛋白质动力学仍然是一个主要的挑战。在这里，我们描述了一种基于全内反射荧光显微镜(TIRFM)成像的自动化、单分子协议，允许在活的植物细胞中进行蛋白质跟踪和亚基计数。该协议使用TIRFM对表达定位于质膜的荧光标记蛋白的转基因植物组织进行成像。接下来，跟踪算法量化荧光蛋白质运动类型、临时颗粒位移和蛋白质光漂白步骤的动态变化。这一方案使研究人员能够研究异质分布蛋白质的动力学特征。这种方法在利用TIRFM或其他荧光成像技术显示的植物活细胞和其他生物标本中各种质膜和细胞内蛋白质的蛋白质动力学和亚单位化学计量学研究中具有潜在的应用价值。整个方案可以在5-6小时内完成。
Measuring the mobility and interactions of proteins is key to understanding cellular signaling mechanisms; however, quantitative analysis of protein dynamics in living plant cells remains a major challenge. Here we describe an automated, single-molecule protocol based on total internal reflection fluorescence microscopy (TIRFM) imaging that allows protein tracking and subunit counting in living plant cells. This protocol uses TIRFM to image transgenic plant tissues expressing fluorescently tagged proteins that are localized to the plasma membrane. Next, a tracking algorithm quantifies dynamic changes in fluorescent protein motion types, temporary particle displacement and protein photobleaching steps. This protocol allows researchers to study the kinetic characteristics of heterogeneously distributed proteins. The approach has potential applications for studies of protein dynamics and subunit stoichiometry for a wide variety of plasma membrane and intracellular proteins in living plant cells and other biological specimens visualized by TIRFM or other fluorescence imaging techniques. The whole protocol can be completed in 5–6 h.