Fluorescently-Labeled Estradiol Internalization and Membrane Trafficking in Live N-38 Neuronal Cells Visualized with Total Internal Reflection Fluorescence Microscopy.
Fluorescently-Labeled Estradiol Internalization and Membrane Trafficking in Live N-38 Neuronal Cells Visualized with Total Internal Reflection Fluorescence Microscopy.
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DOI：
10.4172/2157-7536.s12-002
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发表时间：
2013-04-20
期刊：
影响因子：
--
通讯作者：
Dominguez R
中科院分区：
文献类型：
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作者：
Kisler K;Chow RH;Dominguez R
雌二醇是一种结合并激活雌二醇受体的类固醇激素。已知这些受体的激活可调节神经元生理学并提供神经保护，但尚未完全了解雌二醇如何介导神经系统的这些作用。雌二醇受体-α（ERα）亚群的激活最初被鉴定为一种核蛋白，定位于质膜，似乎是对脑损伤和疾病的神经保护的关键步骤。以前，我们表明，雌二醇刺激下丘脑原代神经元细胞膜ERα的快速和短暂的运输，和膜不渗透雌二醇（E6 BSA-FITC）内化到皮质神经元内体在体外。这些发现支持雌二醇通过触发内吞作用激活和下调质膜ERα的概念。在这里，我们使用TIRFM（全内反射荧光显微镜）成像的贩运E6 BSA-FITC，和GFP标记的ERα，在活细胞中的真实的时间。我们发现，E6 BSA-FITC激活质膜ER导致在活的N-38神经元，永生化下丘脑细胞系的荧光配体的内化。用ER拮抗剂ICI 182，780预处理可减少观察到的E6 BSA-FITC标记斑点的数量。我们还在活的N-38神经元中观察到E6 BSA-FITC与标记内体和溶酶体的FM 4 -64和LysoTracker荧光染料共定位。我们的结果进一步证明了质膜ERα激活会导致受体的内吞作用。
Estradiol is a steroid hormone that binds and activates estradiol receptors. Activation of these receptors is known to modulate neuronal physiology and provide neuroprotection, but it is not completely understood how estradiol mediates these actions on the nervous system. Activation of a sub-population of estradiol receptor-α (ERα), originally identified as a nuclear protein, localizes to the plasma membrane and appears to be a critical step in neuroprotection against brain injury and disease. Previously we showed that estradiol stimulates the rapid and transient trafficking of plasma membrane ERα in primary hypothalamic neurons, and internalization of membrane-impermeant estradiol (E6BSA-FITC) into cortical neuron endosomes in vitro. These findings support the concept that estradiol activates and down-regulates plasma membrane ERα by triggering endocytosis. Here, we use TIRFM (total internal reflection fluorescence microscopy) to image the trafficking of E6BSA-FITC, and GFP-labeled ERα, in live cells in real time. We show that activation of plasma membrane ERs by E6BSA-FITC result in internalization of the fluorescent ligand in live N-38 neurons, an immortalized hypothalamic cell line. Pretreatment with ER antagonist ICI 182,780 decreased the number of E6BSA-FITC labeled puncta observed. We also observed in live N-38 neurons that E6BSA-FITC co-localized with FM4-64 and LysoTracker fluorescent dyes that label endosomes and lysosomes. Our results provide further evidence that plasma membrane ERα activation results in endocytosis of the receptor.