植物学报 ›› 2023, Vol. 58 ›› Issue (4): 590-601.DOI: 10.11983/CBB22102
罗鹏云1,2,3,4, 钱虹萍1,2,3,4, 刘艳5, 徐昌文1,2,3,4, 崔亚宁1,2,3,4(
)
首页收稿日期:2022-06-06
接受日期:2022-11-12
出版日期:2023-07-01
发布日期:2022-11-29
通讯作者:
*E-mail: cuiyaning@bjfu.edu.cn
基金资助:
Pengyun Luo1,2,3,4, Hongping Qian1,2,3,4, Yan Liu5, Changwen Xu1,2,3,4, Yaning Cui1,2,3,4()
Received:2022-06-06
Accepted:2022-11-12
Online:2023-07-01
Published:2022-11-29
Contact:
*E-mail: cuiyaning@bjfu.edu.cn
摘要： 质膜蛋白是细胞膜的重要组分之一, 在细胞的物质转运、离子交换、信号转导以及代谢过程中起着重要作用。质膜蛋白在膜上是运动的, 生长发育和环境因素均可改变其运动方式。因此, 研究影响质膜蛋白运动的因素及调控机制对于理解植物生长发育和应对环境改变至关重要。近年来, 显微技术发展迅速, 使得关于质膜蛋白动力学调控机制的研究逐步深入。该文详细介绍了质膜蛋白动力学及其影响因素, 概述了近几年在质膜蛋白动力学研究中常用的显微成像技术, 以期为深入研究质膜蛋白的生物学功能提供参考。
罗鹏云, 钱虹萍, 刘艳, 徐昌文, 崔亚宁. 质膜蛋白动力学的调控及其研究方法. 植物学报, 2023, 58(4): 590-601.
Pengyun Luo, Hongping Qian, Yan Liu, Changwen Xu, Yaning Cui. Regulation of Plasma Membrane Protein Dynamics and Its Research Methods. Chinese Bulletin of Botany, 2023, 58(4): 590-601.
图1 质膜蛋白动力学影响因素 (A) 正常条件下, 质膜蛋白在膜上运动; (B) 纤维素合成抑制剂处理改变质膜蛋白的横向扩散; (C) 微丝/微管抑制剂处理改变质膜蛋白的横向扩散; (D) MβCD处理改变质膜蛋白的横向扩散。
Figure 1 Factors affecting the dynamics of plasma membrane protein (A) Plasma membrane proteins are relatively stationary on the membrane under normal conditions; (B) The lateral diffusion of plasma membrane proteins was changed when the cell wall was damaged by cellulose synthesis inhibitors; (C) Microfilament/microtubule inhibitor treatment disrupts the cytoskeleton and changes the lateral diffusion movement of plasma membrane proteins; (D) MβCD treatment destroys the membrane raft and changes the lateral diffusion of plasma membrane proteins.
图2 质膜蛋白动力学技术 (A) 由折射到全内反射(total internal reflection, TIR): 1束光在2种不同的介质中传播, 介质的折射率分别为n1和n2, 入射角为α, 由Snell定律可知, 光会发生折射进入另一种介质, 且在另一种介质中的传播角度与原介质不同, 当入射角α1等于甚至大于临界角α2时, 光就不会进入到较小折射率的介质中, 而是完全被反射到原来的介质中, 即发生全内反射; (B) 在荧光相关光谱法(FCS)中, 单个标记分子在焦体积内外的扩散会引起荧光随时间的波动, 通过将荧光涨落曲线拟合到适当的扩散模型, 可计算粒子数及其扩散系数; (C) 选择感兴趣区域(ROI), 用强激光束漂白, 并测量ROI中的荧光恢复时间; (D) 超分辨显微成像技术的原理示意图。FCS、SIM、STED、PALM和STORM同表1。
Figure 2 Technology of plasma membrane protein dynamics (A) From refraction to total internal reflection (TIR): the incident beam approaching the interface between two media with different indices of refraction n1 and n2 at an angle α will be refracted according to Snell’s law, beam propagation after the interface occurs thus at a different angle, if the incident angle α1 is greater than or equal to the critical angle α2, the beam does not propagate in the optically less-dense medium, but instead undergoes total reflection at the interface; (B) The diffusion of single labeled molecules in and out of the focal volume in fluorescence correlation spectroscopy (FCS) induces fluctuations in fluorescence with time, the number of particles and their diffusion coefficients can be calculated by fitting the fluorescence fluctuation curve to an adequate diffusion model; (C) A region of interest (ROI) is selected bleached with an intense laser beam, and the fluorescence recovery in the ROI is measured over time; (D) Schematic diagram of the principles of super-resolution microscopic imaging techniques. FCS, SIM, STED, PALM and STORM see Table 1.
| 技术名称 | 适用对象 | 优点 | 缺点 |
|---|---|---|---|
| TIRFM | 膜蛋白和脂类等分子 | 只激发靠近界面100-200 nm处的荧光分子, 信噪比显著提高 | 成像受衍射极限的限制, 且轴向分辨率局限于宽场范围 |
| FCS | 可自由扩散的荧光分子 | 灵敏度高, 空间分辨率高, 非侵入性检测等 | 不适用于高浓度和扩散速度慢的样本, 高激发功率下荧光团会出现光漂白 |
| FRAP | 活细胞内分子 | 可用于功能性测量活细胞中的蛋白质迁移率 | 只能检测荧光分子的群体移动, 不能观察单个分子的移动 |
| SIM | 活细胞内分子 | 成像速度快, 普适性强, 无须特殊的染料标记 | 硬件装置昂贵, 成像时对光条件、原始数据的质量和数量要求严苛, 捕捉动态性较高的目标时会出现引入动态伪影的问题, 对活细胞内动态性较高的亚细胞结构长时程成像存在明显的局限性 |
| STED | 活体生物样品 | 荧光探针无光开关的特殊要求, 时间分辨率高 | |
| PALM | 细胞外源表达的荧光蛋白 | 可用于活细胞测量, 分辨率可达1 nm量级 | |
| STORM | 内源性蛋白物质 | 可研究细胞内源性蛋白的超分辨率定位 |
表1 质膜蛋白动力学研究方法的比较
Table 1 Comparison of methods on plasma membrane protein dynamics research
| 技术名称 | 适用对象 | 优点 | 缺点 |
|---|---|---|---|
| TIRFM | 膜蛋白和脂类等分子 | 只激发靠近界面100-200 nm处的荧光分子, 信噪比显著提高 | 成像受衍射极限的限制, 且轴向分辨率局限于宽场范围 |
| FCS | 可自由扩散的荧光分子 | 灵敏度高, 空间分辨率高, 非侵入性检测等 | 不适用于高浓度和扩散速度慢的样本, 高激发功率下荧光团会出现光漂白 |
| FRAP | 活细胞内分子 | 可用于功能性测量活细胞中的蛋白质迁移率 | 只能检测荧光分子的群体移动, 不能观察单个分子的移动 |
| SIM | 活细胞内分子 | 成像速度快, 普适性强, 无须特殊的染料标记 | 硬件装置昂贵, 成像时对光条件、原始数据的质量和数量要求严苛, 捕捉动态性较高的目标时会出现引入动态伪影的问题, 对活细胞内动态性较高的亚细胞结构长时程成像存在明显的局限性 |
| STED | 活体生物样品 | 荧光探针无光开关的特殊要求, 时间分辨率高 | |
| PALM | 细胞外源表达的荧光蛋白 | 可用于活细胞测量, 分辨率可达1 nm量级 | |
| STORM | 内源性蛋白物质 | 可研究细胞内源性蛋白的超分辨率定位 |
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