庆祝荧光相关光谱（FCS） 50周年：利用光稳定性gfp， mStayGold和StayGold/E138D推进活细胞大规模平行FCS研究

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. General subjects Pub Date : 2025-04-17 DOI:10.1016/j.bbagen.2025.130809

Sho Oasa , Borislav Stoyanov , Yuta Hamada , Stanko N. Nikolić , Aleksandar J. Krmpot , Akira Kitamura , Vladana Vukojević

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引用次数: 0

摘要

50多年来，荧光相关光谱（FCS）仍然是定量表征分子细胞动力学及其在活细胞中的浓度和相互作用的基础技术。增强型绿色荧光蛋白（eGFP）因其亮度、光稳定性和缺乏翻译后修饰而长期以来一直是活细胞FCS的首选标签。然而，较低的eGFP光稳定性限制了测量持续时间，为研究需要较长测量时间的动态细胞过程带来了挑战。荧光蛋白工程的最新进展已经产生了mStayGold和StayGold/E138D，这是两种高度光稳定性的单体GFP变体。在本研究中，我们通过量化活细胞中糖皮质激素受体（GR）同二聚化和核输入/输出动态来评估它们在活细胞中的表现和FCS的效用。我们的研究表明，mStayGold和StayGold/E138D的亮度是eGFP的两倍，显著提高了信噪比（SNR）。利用大规模平行FCS （mpFCS）和双焦点相互关联来表征GR核细胞质沿核膜的运输方向，我们也证实这些蛋白比eGFP表现出显著改善的光稳定性。

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Celebrating 50 years of fluorescence correlation spectroscopy (FCS): Advancing live-cell massively parallel FCS studies with photostable GFPs, mStayGold and StayGold/E138D

More than 50 years after its inception, fluorescence correlation spectroscopy (FCS) remains a cornerstone technique for quantitative characterization of the cellular dynamics of molecules and their concentration and interactions in live cells. The enhanced green fluorescent protein (eGFP) has long been a preferred tag in live-cell FCS, valued for its brightness, photostability and lack of posttranslational modifications. However, low eGFP photostability limits measurement durations, posing challenges for studying dynamic cellular processes necessitating longer measurement time. Recent advancements in fluorescent protein engineering have yielded mStayGold and StayGold/E138D, two highly photostable monomeric GFP variants. In this study, we evaluate their performance in live cells and utility for FCS by quantifying glucocorticoid receptor (GR) homodimerization and nuclear import/export dynamics in live cells. Our study shows that both mStayGold and StayGold/E138D exhibit twice the brightness of eGFP, significantly enhancing the signal-to-noise ratio (SNR). Using massively parallel FCS (mpFCS) and two-foci cross-correlation to characterize the direction of GR nucleocytoplasmic transport along the nuclear envelope, we also confirm that these proteins show significantly improved photostability over eGFP.

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来源期刊

Biochimica et biophysica acta. General subjects 生物-生化与分子生物学

CiteScore

6.40

自引率

0.00%

发文量

139

审稿时长

30 days

期刊介绍： BBA General Subjects accepts for submission either original, hypothesis-driven studies or reviews covering subjects in biochemistry and biophysics that are considered to have general interest for a wide audience. Manuscripts with interdisciplinary approaches are especially encouraged.