通过受控脂质交换和成像荧光相关光谱评估活细胞中的跨膜功能耦合。

4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-25 DOI:10.1016/bs.mie.2024.04.001
Arpita Tripathy, Sudipti Priyadarsinee, Nirmalya Bag
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引用次数: 0

摘要

内叶和外叶之间的生物物理耦合(称为叶间耦合或跨膜耦合)是哺乳动物活细胞质膜的基本组织原理。有人认为,两个小叶之间基于脂质的相互作用是跨膜耦合的主要机制。然而,只有少数实验证据支持活细胞中存在这种相互作用。这似乎是由于缺乏实验策略来扰乱一个小叶的脂质成分,以及缺乏定量技术来评估对侧小叶的生物物理特性。现有的策略通常依赖于在其中一片叶子中固定和聚集一种成分,以及技术要求较高的生物物理工具来评估对另一片叶子的影响。近年来,伦敦研究小组开发了一种简单而优雅的方法,即甲基-α-环糊精催化的脂质交换(LEX),可有效地将小叶外层脂质与所选择的外源脂质进行交换。在这里,我们采用这种方法来扰乱外小叶脂质的组成。通过比较未扰动和扰动条件下内叶局部脂质探针的扩散情况,评估内叶的相应变化。我们采用了高度复用的成像荧光相关光谱法(ImFCS),通过配备快速灵敏相机的商用或自制全内反射荧光显微镜来测定脂质探针的扩散系数。结合使用 LEX 和 ImFCS,我们直接展示了基于脂质的跨膜耦合,无需固定静息状态下活体肥大细胞中的膜成分。总之,我们提出了一种相对简单的实验策略来定量评估活细胞中的跨膜耦合。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Evaluation of functional transbilayer coupling in live cells by controlled lipid exchange and imaging fluorescence correlation spectroscopy.

Biophysical coupling between the inner and outer leaflets, known as inter-leaflet or transbilayer coupling, is a fundamental organizational principle in the plasma membranes of live mammalian cells. Lipid-based interactions between the two leaflets are proposed to be a primary mechanism underlying transbilayer coupling. However, there are only a few experimental evidence supporting the existence of such interactions in live cells. This is seemingly due to the lack of experimental strategies to perturb the lipid composition in one leaflet and quantitative techniques to evaluate the biophysical properties of the opposite leaflet. The existing strategies often dependent on immobilization and clustering a component in one of the leaflets and technically demanding biophysical tools to evaluate the effects on the opposing leaflet. In the recent years, the London group developed a simple but elegant method, namely methyl-alpha-cyclodextrin catalyzed lipid exchange (LEX), to efficiently exchange outer leaflet lipids with an exogenous lipid of choice. Here, we adopted this method to perturb outer leaflet lipid composition. The corresponding changes in the inner leaflet is evaluated by comparing the diffusion of lipid probes localized in this leaflet in unperturbed and perturbed conditions. We employed highly multiplexed imaging fluorescence correlation spectroscopy (ImFCS), realized in a commercially available or home-built total internal reflection fluorescence microsocope equipped with a fast and sensitive camera, to determine diffusion coefficient of the lipid probes. Using the combination of LEX and ImFCS, we directly demonstrate lipid-based transbilayer coupling that does not require immobilization of membrane components in live mast cells in resting conditions. Overall, we present a relatively straightforward experimental strategy to evaluate transbilayer coupling quantitively in live cells.

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来源期刊
Methods in enzymology
Methods in enzymology 生物-生化研究方法
CiteScore
2.90
自引率
0.00%
发文量
308
审稿时长
3-6 weeks
期刊介绍: The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.
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