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Fluorescent Probes for Lipid Membranes: From the Cell Surface to Organelles
Accounts of Chemical Research ( IF 18.3 ) Pub Date : 2022-12-19 , DOI: 10.1021/acs.accounts.2c00586
Andrey S Klymchenko 1
Affiliation  

Biomembranes are ubiquitous lipid structures that delimit the cell surface and organelles and operate as platforms for a multitude of biomolecular processes. The development of chemical tools─fluorescent probes─for the sensing and imaging of biomembranes is a rapidly growing research direction, stimulated by a high demand from cell biologists and biophysicists. This Account focuses on advances in these smart molecules, providing a voyage from the cell frontier─plasma membranes (PM)─toward intracellular membrane compartments─organelles. General classification of the membrane probes can be based on targeting principles, sensing profile, and optical response. Probes for PM and organelle membranes are designed based on multiple targeting principles: conjugation with natural lipids or synthetic targeting ligands and in situ cell labeling by bio-orthogonal chemistry, conjugation to protein tags, and receptor–ligand interactions. Thus, to obtain membrane probes targeting PM with selectivity to one leaflet, we designed membrane anchor ligands based on a charged group and an alkyl chain. According to the sensing profile, we define basic membrane markers with constant emission and probes for biophysical and chemical sensing. The markers are built from classical fluorophores, exemplified by a series of bright cyanines and BODIPY dyes bearing the PM anchors (MemBright). Membrane probes for biophysical sensing are based on environment-sensitive fluorophores: (1) polarity-sensitive solvatochromic dyes; (2) viscosity-sensitive fluorescent molecular rotors; (3) mechanosensitive fluorescent flippers; and (4) voltage-sensitive electrochromic dyes. Our solvatochromic probes based on Nile Red (NR12S, NR12A, NR4A), Laurdan (Pro12A), and 3-hydroxyflavone (F2N12S) through polarity-sensing can visualize liquid ordered and disordered phases of lipid membranes, sense lipid order and its heterogeneity in cell PM, detect apoptosis, etc. Chemically sensitive probes, combining a dye, membrane-targeting ligand, and molecular recognition unit, enable the detection of pH, ions, redox species, lipids, and proteins at the biomembrane surface. In terms of the optical response profile, we can identify (1) fluorogenic (turn-on) probes, allowing background-free imaging; (2) ratiometric probes, e.g., solvatochromic probes, which enable ratiometric imaging by changing their emission/excitation color; (3) fluorescence lifetime-responsive probes, e.g., fluorescence molecular rotors and flippers, suitable for fluorescence lifetime imaging (FLIM); and (4) switchable probes, important for single-molecule localization microscopy. We showed that combining solvatochromic probes with on–off switching through a reversible binding specifically to cell PM enables the mapping of their biophysical properties with superior resolution. While the majority of efforts have been focused on PM, the probes for cellular organelles, such as endoplasmic reticulum, mitochondria, Golgi apparatus, etc., emerge rapidly. Thus, nontargeted solvatochromic probes can distinguish organelles by the emission color. Targeted solvatochromic probes based on Nile Red revealed unique signatures of polarity and lipid order of individual organelles and their different sensitivities to oxidative or mechanical stress. Lipid droplets, which are membraneless lipidic structures, constitute another interesting organelle target for probing the cell stress. Currently, we stand at the beginning of a long route with big challenges ahead, in particular (1) to achieve superior organelle specificity; (2) to label specific biomembrane leaflets, notably the inner leaflet of PM; (3) to detect lipid organization in a proximity of specific proteins; and (4) to probe biomembranes in tissues and animals.

中文翻译:

脂质膜荧光探针:从细胞表面到细胞器

生物膜是普遍存在的脂质结构,它们界定细胞表面和细胞器,并作为多种生物分子过程的平台。在细胞生物学家和生物物理学家的高需求刺激下,开发用于生物膜传感和成像的化学工具(荧光探针)是一个快速发展的研究方向。本帐户重点关注这些智能分子的进展,提供从细胞边界──质膜 (PM)──到细胞内膜区室──细胞器的航行。膜探针的一般分类可以基于靶向原理传感特性光学响应。. 用于 PM 和细胞器膜的探针是根据多种靶向原理设计的:与天然脂质合成靶向配体结合,通过生物正交化学进行原位细胞标记与蛋白质标签结合,以及受体-配体相互作用。因此,为了获得对单张具有选择性的靶向 PM 的膜探针,我们设计了基于带电基团和烷基链的膜锚配体。根据传感概况,我们定义了具有恒定发射的基本膜标记用于生物物理和化学传感的探针。标记由经典荧光团构建, 以一系列带有 PM 锚 (MemBright) 的明亮花青和 BODIPY 染料为例。用于生物物理传感的膜探针基于环境敏感的荧光团:(1)极性敏感的溶剂化显色染料;(2) 粘敏荧光分子转子;(3) 机械敏感荧光脚蹼;(4)电压敏感电致变色染料。我们基于尼罗红 (NR12S、NR12A、NR4A)、劳尔丹 (Pro12A) 和 3-羟基黄酮 (F2N12S) 的溶剂化显色探针通过极性感应可以可视化脂质膜的液体有序相和无序相,感知脂质顺序及其在细胞中的异质性PM、检测细胞凋亡等 C化学敏感探针结合染料、膜靶向配体和分子识别单元,能够检测生物膜表面的 pH 值、离子、氧化还原物质、脂质和蛋白质。就光学响应曲线而言,我们可以识别 (1)荧光(开启)探针,允许无背景成像;(2)比例探针例如溶剂化显色探针,通过改变它们的发射/激发颜色来实现比例成像;(3)荧光寿命响应探针例如荧光分子转子和鳍状肢,适用于荧光寿命成像(FLIM);(4)可切换探头,对单分子定位显微镜很重要。我们表明,通过与细胞 PM 特异性可逆结合,将溶剂化显色探针与开-关切换相结合,能够以更高的分辨率绘制它们的生物物理特性。虽然大部分努力都集中在 PM 上,但内质网、线粒体、高尔基体等细胞器的探针迅速出现。因此,非靶向溶剂化显色探针可以通过发射颜色区分细胞器。基于尼罗红的靶向溶剂化显色探针揭示了单个细胞器的极性和脂质顺序的独特特征,以及它们对氧化或机械应力的不同敏感性。脂滴是无膜脂质结构,构成了另一个有趣的细胞器靶标,用于探测细胞应激。目前,我们正站在一条漫长道路的开端,面临着巨大的挑战,特别是 (1) 实现卓越的细胞器特异性;(2) 标记特定的生物膜小叶,特别是 PM 的内小叶;(3) 检测特定蛋白质附近的脂质组织;(4) 探测组织和动物的生物膜。
更新日期:2022-12-19
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