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植物学报 >

2025 , Vol. 60 >Issue 1: 1 - 0

DOI: https://doi.org/10.11983/CBB24040

技术方法

植物细胞质膜有序性的活细胞定量分析

  • 陈秀秀 ,
  • 唐玲 ,
  • 胡文佳 ,
  • 杨照麟 ,
  • 邓馨 ,
  • 王晓华
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  • 1中国科学院植物研究所, 植物多样性与特色经济作物重点实验室, 北京 100093; 2国家植物园, 北京 100093; 3中国科学院大学, 北京 100049

收稿日期: 2024-03-12

  修回日期: 2024-05-11

  网络出版日期: 2024-05-30

基金资助

北京市自然科学基金(No.5222021)和国家自然科学基金(No.32170412)

收起

Quantitative Analysis of Plasma Membrane Order in Live Plant Cells

  • CHEN Xiu-Xiu ,
  • TANG Ling ,
  • HU Wen-Jia ,
  • YANG Zhao-Lin ,
  • DENG Xin ,
  • YU Xiao-Hua
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  • 1State Key Laboratory of Plant Diversity and Special Crops, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China; 2China National Botanical Garden, Beijing 100093, China; 3University of Chinese Academy of Sciences, Beijing 100049, China

Received date: 2024-03-12

  Revised date: 2024-05-11

  Online published: 2024-05-30

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摘要

质膜微区是细胞质膜上富含甾醇和鞘磷脂的微结构域, 参与众多生物学过程, 如信号转导、囊泡转运、胞吞和胞吐, 因此质膜微区动态过程是植物细胞生物学研究的重要领域之一。利用荧光探针结合荧光显微镜已广泛应用于检测植物活细胞状态。PA (push-pull pyrene)是一种基于芘的新型高效稳定的荧光探针, 但其应用于植物活细胞成像研究极少。我们利用PA探针和激光共聚焦显微镜技术, 结合图像处理和极性归一化数值作图法对拟南芥(Arabidopsis thaliana)根中活细胞质膜有序度进行定量分析。结果发现, PA探针在拟南芥根细胞质膜中液态有序相的发射光谱为500–550 nm, 液态无序相的发射光谱为580–700 nm。甾醇抽提剂MβCD处理野生型拟南芥导致质膜有序度降低。在甾醇合成关键的甲基转移酶双突变体smt2/smt3中, 其质膜的有序度与野生型株系经甾醇抽提剂MβCD处理后的有序度结果一致。smt2/smt3突变体中, 根毛细胞质膜的有序度低于野生型根毛细胞质膜的有序度, 表明甾醇作为膜微区关键组分对调节质膜的有序度发挥了重要作用。本研究为检测植物活细胞质膜动力学特征和质膜微区的变化, 提供了一种直观和快速的检测手段。

本文引用格式

陈秀秀 , 唐玲 , 胡文佳 , 杨照麟 , 邓馨 , 王晓华 . 植物细胞质膜有序性的活细胞定量分析[J]. 植物学报, 2025 , 60(1) : 1 -0 . DOI: 10.11983/CBB24040

Abstract

Membrane microdomains, which are highly dynamic structures rich in sterols and sphingolipids on the plasma membrane, play a crucial role in various biological processes such as signal transduction, vesicle transport, endocytosis, and exocytosis. Consequently, the investigation of membrane microdomain dynamics stands as one of the important areas of research in plant cell biology. Fluorescence probes combined with fluorescence microscopy are widely used to monitor the status of living plant cells. PA probe (push-pull pyrene) is a novel, highly efficient and stable fluorescence probe based on pyrene, however, its application in imaging studies of living plant cells is limited. In this study, we used PA probes and Laser scanning confocal microscopy, combined with image processing and polar normalized value mapping method, to quantitatively analyze the order of the plasma membrane in Arabidopsis root cells. The results showed that the emission spectrum of the liquid-ordered phase in the plasma membrane of Arabidopsis root cells labelled by the PA probe was 500-550 nm, while the emission spectrum of the liquid-disordered phase was 580-700 nm. Treatment of wild-type plants with the sterol extraction agent MβCD resulted in a decrease in plasma membrane order. In the double mutant smt2/smt3 lacking the key methyltransferase in sterol synthesis, the plasma membrane order was consistent with that of the wild-type plants after treatment with MβCD. In the smt2/smt3 mutant, the plasma membrane order of the root hair cells was lower than that of the plasma membrane order of wild-type root hair cell, indicating that sterols, as key components of membrane microdomains, play an important role in regulating the order of the plasma membrane. This study provides a straightforward and rapid detection method for monitoring the dynamic characteristics of living plant cell membrane and changes in membrane microdomains.

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参考文献

董紫怡，宋程威，崔亚宁 (2019). 膜微区相关结构模型及甾醇成像技术的研究进展. 电子显微学报 38, 542-549. 黄鑫华, 刘伟, 田世平 陈彤 (2023). 蛋白液-液相分离调控植物发育及胁迫应答研究进展. 植物学报 58, 946-955. 罗鹏云, 钱虹萍, 刘艳, 徐昌文 崔亚宁 (2023). 质膜蛋白动力学的调控及其研究方法. 植物学报 58, 590-601. 吕雪芹, 金柯, 刘家恒, 崔世修, 李江华, 堵国成, 刘龙 (2021). 工业模式微生物膜有序性的活细胞定量分析. 中国生物工程杂志41, 20-29. 严旭, 徐梅, 王玉同, 潘伟槐, 潘建伟, 寿建昕, 王超 (2022). 植物胞吞和胞吐的耦合调控. 植物学报57, 375-387. 赵晓玉 (2014). 新型荧光探针di-4-ANEPPDHQ在拟南芥质膜微区显微成像和定量检测中的应用. 硕士论文. 北京: 北京林业大学. pp. 1-82. 左春山, 刘大勇, 徐启杰, 时文中, 牛静, Chass GC (2013). 植物甾醇的结构与功能的研究进展. 河南科技 9, 211-213. Brown DA (1992). Interactions between gpi-anchored proteins and membrane lipids. Trends Cell Biol 2, 338-343. Brown DA, London E (2000). Structure and Function of Sphingolipid- and Cholesterol-rich Membrane Rafts. J Biol Chem 275, 17221-17224 Carland F, Nelson, FT (2010). The sterol methyltransferases smt1, smt2, and smt3 influence Arabidopsis development through nonbrassinosteroid products. Plant Physiol 153, 741-756. Carland FM (2002). The identification of cvp1 reveals a role for sterols in vascular patterning. Plant Cell 14, 2045-2058. Chiantia S, Ries J, Kahya N, Schwille P (2010). Combined afm and two-focus sfcs study of raft-exhibiting model membranes. Chemphyschem 7, 2409-2418. Filippov A, Greger O, G?ran L (2003). The effect of cholesterol on the lateral diffusion of phospholipids in oriented bilayers. Biophys J 84, 3079-3086. Gerke V, Gavins FNE, Geisow M, Grewal T, Jaiswal JK, Nylandsted J, Rescher U (2024). Annexins-a family of proteins with distinctive tastes for cell signaling and membrane dynamics. Nat Commun 15, 1574. Goksu EI, Vanegas JM, Blanchette CD, Lin WC, Longo ML (2009). AFM for structure and dynamics of biomembranes. BBA Rev Biomembranes 1788, 254-266. Hoppe T, Rape M, Jentsch S (2001). Membrane-bound transcription factors: regulated release by RIP or RUP. Curr Opin Cell Biol 13, 344-348. Jin L, Millard AC, Wuskell JP, Clark HA, Loew LM (2005). Cholesterol-enriched lipid domains can be visualized by di-4-ANEPPDHQ with linear and nonlinear optics. Biophys J 89, L04-06. Kusumi A, Fujiwara TK, Chadda R, Xie M, Tsunoyama TA, Kalay Z, Kasai RS, Suzuki KGN (2012). Dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of singer and nicolson's fluid-mosaic model. Annu Rev Cell Dev Biol 28, 215-250. Lingwood D, Simons, K (2010). Lipid rafts as a membrane-organizing principle. Science 327, 46-50. Mesmin B, Bigay J, Polidori J, Jamecna D, Lacas GS, Antonny B (2017). Sterol transfer, PI4P consumption, and control of membrane lipid order by endogenous osbp. Embo J 36, 3156-3174. Munro S (2003). Lipid rafts: elusive or illusive? Cell 115, 377-388. Niko Y, Didier P, Mely Y, Konishi GI, Klymchenko AS (2016). Bright and photostable push-pull pyrene dye visualizes lipid order variation between plasma and intracellular membranes. Sci Rep 6, 18870. Niko Y, Kawauchi S, Konishi G (2013). Solvatochromic pyrene analogues of prodan exhibiting extremely high fluorescence quantum yields in apolar and polar solvents. Chemistry 19, 9760-9765. Roche，Gerbeau P，Buhot，Thomas，Bonneau，Gresti，Mongrand，Perrier C，Simon-Plas JM (2008). Depletion of phytosterols from the plant plasm a membrane provides evidence for disruption of lipid rafts. Faseb J 22, 3980-3991. Sergey I, Maria JH (2024). Receptor-associated kinases control the lipid provisioning program in plant-fungal symbiosis. Science 383, 443-448. Sezgin E, Sadowski T, Simons K (2014). Measuring Lipid Packing of Model and Cellular Membranes with Environment Sensitive Probes. Langmuir 30, 8160-8166. Shaw JE, Epand RF, Epand RM, Li Z, Bittman R, Yip CM (2006). Correlated fluorescence-atomic force microscopy of membrane domains: structure of fluorescence probes determines lipid localization. Biophys J 90, 2170-2178. Simons K (2000). Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1, 31-41. Simons K, Ikonen E (1997). Functional rafts in cell membranes. Nature 387, 569-572. Singer SJ, Nicolson GL (1972). The Fluid Mosaic Model of the Structure of Cell Membranes. Science, 175, 720-731. Parasassi T, Gratton E, Yu WM, Wilson P, Levi M (1997). Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes. Biophys J 72, 2413-2429. Tang L, Li Y, Zhong C, Deng X, Wang X (2021). Plant Sterol Clustering Correlates with Membrane Microdomains as Revealed by Optical and Computational Microscopy. Membranes 11, 747. Zhang L, Xing J, Lin J (2019). At the intersection of exocytosis and endocytosis in plants. New Phytol 224, 1479-1489.
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