| Live Cell FRET Analysis of the Conformational Changes of Human P-glycoprotein
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Author:
Date:
2021-02-20
[Abstract] The molecular mechanisms of P-glycoprotein (P-gp; also known as MDR1 or ABCB1) have been mainly investigated using artificial membranes such as lipid-detergent mixed micelles, artificial lipid bilayers, and membrane vesicles derived from cultured cells. Although these in vitro experiments help illustrate details about the molecular mechanisms of P-gp, they do not reflect physiological membrane environments in terms of lateral pressure, curvature, constituent lipid species, etc. The protocol presented here includes a detailed guide for analyzing the conformational change of human P-gp in living HEK293 cells by using intramolecular fluorescence resonance energy transfer (FRET), in which excitation of the donor fluorophore is transferred to the acceptor without emission of a photon when two ...
[摘要] [摘要] P-糖蛋白（P-gp；也称为MDR1或ABCB1）的分子机制已主要使用人造膜进行研究，例如脂质去污剂混合胶束，人造脂质双层和源自培养细胞的膜囊泡。尽管这些体外实验有助于阐明有关P-gp分子机制的细节，但它们在侧向压力，曲率，脂质成分等方面并未反映出生理膜环境。 此处提供的协议包括一个详细的指南，该指南用于通过使用分子内荧光共振能量转移（FRET）分析活HEK293细胞中人P-gp的构象变化，其中供体荧光团的激发被转移到受体上而不发射光子当两个荧光蛋白非常接近时。将FRET分析与膜通透性相结合，可以在活细胞中评估小分子（如核苷酸）对构象变化的贡献。
[背景] P-糖蛋白（P-gp）的是ATP驱动药转运该压出各种疏水有毒化合物到细胞外空间。P-gp由形成底物转运途径的两个跨膜结构域（TMD）和结合并水解ATP的两个核苷酸结合结构域（NBD）组成。传输至少需要两个P-gp状态。在向内（药物转运前）构型中，两个NBD分开，两个TMD向细胞内侧开放；在向外（药物转运）构象中，NBD是二聚体的，而TMD在细胞外侧略微开放（Kodan et al。，2020 ）。自从发现P-gp （Juliano和Ling，1976； Chen等，1986； Ueda等，1986 ...
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| Spatial Image Correlation Spectroscopy (ICS): A Technique for Average Size Determination of Subcellular Accumulated Structures from Fluorescence Microscopic Images
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Author:
Date:
2020-05-20
[Abstract] Size determination of subcellular structures such as inclusion bodies (IBs) and granules from fluorescent images is important for identification and structural characterization. However, it is often time-consuming just for the comparison of the average size of the structures. Here, we introduce a high-throughput procedure to represent the average size of structures in fluorescent images using Spatial Image Correlation Spectroscopy (SICS). This procedure provides an easier comparison of bodies and granular structures such as inclusion bodies (IBs) including misfolded protein aggregation, granules containing RNA (e.g., stress granules and processing bodies).
[摘要] [摘要] 从荧光图像中确定亚细胞结构（如包涵体（IBs）和颗粒）的尺寸确定对于鉴定和结构表征很重要。然而，它往往是耗费时间只是在结构的平均尺寸的比较。在这里，我们介绍了一种高通量程序，可以使用空间图像相关光谱（SICS）表示荧光图像中结构的平均大小。该程序使物体和颗粒结构的比较容易，例如包含错误折叠的蛋白质聚集的包涵体（IBs），包含RNA的颗粒（例如，应力颗粒和加工体）。
[背景] 包涵体（IBs）经常在神经退行性疾病患者的神经元中，甚至在健康生物体的细胞中也观察到（Ross和Poirier，2004； Anderson和Kedersha，2006； Kitamura 等，2015）。包括错误折叠的蛋白质聚集的IB是神经退行性疾病的标志。确定此类IB的大小对于识别和表征IB非常重要。通常使用荧光蛋白和/或免疫荧光来观察IB和亚细胞结构，因为荧光标记针对感兴趣的结构提供了理想的特异性以对抗背景中的低信号（Kitamura和Kinjo，2018）。为了确定荧光图像中亚细胞结构（如IB）的大小，已使用图像分析软件（例如ImageJ，Fiji，Metamorph 等）直接测量了结构的半径和面积。在此过程中，有必要设计一种能够准确反映特定结构尺寸的遮罩，对于研究人员来说，设计能够正确区分结构边缘的遮罩有时并不容易。ALTH Ô ...
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| Microscopic Observation, Three-dimensional Reconstruction, and Volume Measurements of Sperm Nuclei
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Author:
Date:
2015-04-05
[Abstract] Karyogamy, a migration of the sperm nucleus toward the egg nucleus and their subsequent nuclear fusion, is an important biological event for initiating zygote formation toward early embryogenesis in angiosperms. However, how the male nucleus approaches and fuses with the female nucleus still remains unclear. Recently, time-lapse measurement of nuclear volume during karyogamic events revealed that the sperm nucleus enlarges during contact with the egg nucleus via possible one-directional migration of egg chromatin into sperm nucleus (Ohnishi et al., 2014). Here, we describe the protocol for microscopic observation, three-dimensional reconstruction, and volume measurements of sperm nuclei in rice zygotes/fused gametes, which are produced by an in vitro fertilization system ...
[摘要] 核型，精子核朝向卵核和它们随后的核融合的迁移是引起被子植物中早期胚胎形成的合子形成的重要生物事件。然而，男性核如何接近和融合与女性核仍然不清楚。最近，核染色体事件中核体积的延时测量显示精子核在与卵核接触期间扩大，通过卵染色质可能的一向迁移到精子核中（Ohnishi等人，2014年， ）。在这里，我们描述了用于显微观察，三维重建和体积测量的水稻合子/融合配子中的精子细胞核的方案，其由体外受精系统产生（Uchiumi等人al。，2006; Uchiumi等人，2007）。本协议将适用于监测细胞分裂，分化，去分化和极性形成以及核型进展期间细胞中的核动力学。
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