Mapping the Direction of Nucleocytoplasmic Transport of Glucocorticoid Receptor (GR) in Live Cells Using Two-Foci Cross-Correlation in Massively Parallel Fluorescence Correlation Spectroscopy (mpFCS).
Mapping the Direction of Nucleocytoplasmic Transport of Glucocorticoid Receptor (GR) in Live Cells Using Two-Foci Cross-Correlation in Massively Parallel Fluorescence Correlation Spectroscopy (mpFCS).
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DOI：
10.1021/acs.analchem.3c01427
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发表时间：
2023-10-17
影响因子：
7.4
通讯作者：
Vukojevic, Vladana
中科院分区：
文献类型：
--
作者：
Nikolic, Stanko N.;Oasa, Sho;Krmpot, Aleksandar J.;Terenius, Lars;Belic, Milivoj R.;Rigler, Rudolf;Vukojevic, Vladana
转录因子的核细胞质转运对于正常细胞功能至关重要，其分解是许多疾病的主要促成因素。糖皮质激素受体（GR）是一种进化上保守的配体依赖性转录因子，可调节体内平衡和应激反应，是炎症和癌症治疗的重要靶点。在未受刺激的细胞中，GR 存在于细胞质中，与大型多蛋白复合物中的其他分子结合。在内源性或合成配体刺激后，GR 易位到细胞核，其中 GR 通过直接结合糖皮质激素反应元件或通过与其他转录因子物理结合来调节许多基因的转录。虽然人们对 GR 功能的分子机制了解很多，但 GR 核细胞质运输方向性的空间组织仍然不太清楚，并且尚不清楚 GR 的双向核细胞质流动如何在受刺激的细胞中协调。在这里，我们在大规模并行荧光相关光谱 (mpFCS) 系统中使用两焦点互相关来绘制活细胞中沿着核膜不同位置的 GR 易位的方向性。我们从理论上和实验上证明，当 mpFCS 设置中两个附近的观测体积元素 (OVE) 沿分子运动轨迹定位时，来自两个附近观测体积元素 (OVE) 的信号的互相关会呈现出尖锐的峰值，并且峰值的时间位置对应于分子在两个 OVE 之间的平均飞行时间。因此，可以在沿着核膜的几个位置同时确定核细胞质运输的方向和速度。我们发现，在配体诱导的 GR 易位下，GR 的核细胞质输入/输出同时进行，但在细胞核的不同位置进行。我们的数据表明，mpFCS 可以详细表征活细胞中定向核细胞质运输的异质性，对于旨在了解如何协调大分子通过核孔复合体 (NPC) 的双向流动以避免核内转录因子增加/减弱的研究可能非常有价值。
Nucleocytoplasmic transport of transcription factors is vital for normal cellular function, and its breakdown is a major contributing factor in many diseases. The glucocorticoid receptor (GR) is an evolutionarily conserved, ligand-dependent transcription factor that regulates homeostasis and response to stress and is an important target for therapeutics in inflammation and cancer. In unstimulated cells, the GR resides in the cytoplasm bound to other molecules in a large multiprotein complex. Upon stimulation with endogenous or synthetic ligands, GR translocation to the cell nucleus occurs, where the GR regulates the transcription of numerous genes by direct binding to glucocorticoid response elements or by physically associating with other transcription factors. While much is known about molecular mechanisms underlying GR function, the spatial organization of directionality of GR nucleocytoplasmic transport remains less well characterized, and it is not well understood how the bidirectional nucleocytoplasmic flow of GR is coordinated in stimulated cells. Here, we use two-foci cross-correlation in a massively parallel fluorescence correlation spectroscopy (mpFCS) system to map in live cells the directionality of GR translocation at different positions along the nuclear envelope. We show theoretically and experimentally that cross-correlation of signals from two nearby observation volume elements (OVEs) in an mpFCS setup presents a sharp peak when the OVEs are positioned along the trajectory of molecular motion and that the time position of the peak corresponds to the average time of flight of the molecule between the two OVEs. Hence, the direction and velocity of nucleocytoplasmic transport can be determined simultaneously at several locations along the nuclear envelope. We reveal that under ligand-induced GR translocation, nucleocytoplasmic import/export of GR proceeds simultaneously but at different locations in the cell nucleus. Our data show that mpFCS can characterize in detail the heterogeneity of directional nucleocytoplasmic transport in a live cell and may be invaluable for studies aiming to understand how the bidirectional flow of macromolecules through the nuclear pore complex (NPC) is coordinated to avoid intranuclear transcription factor accretion/abatement.
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