| Imaging Microtubules in vitro at High Resolution while Preserving their Structure
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Author:
Date:
2021-04-05
[Abstract] Microtubules (MT) are the most rigid component of the cytoskeleton. Nevertheless, they often appear highly curved in the cellular context and the mechanisms governing their overall shape are poorly understood. Currently, in vitro microtubule analysis relies primarily on electron microscopy for its high resolution and Total Internal Reflection Fluorescence (TIRF) microscopy for its ability to image live fluorescently-labelled microtubules and associated proteins. For three-dimensional analyses of microtubules with micrometer curvatures, we have developed an assay in which MTs are polymerized in vitro from MT seeds adhered to a glass slide in a manner similar to conventional TIRF microscopy protocols. Free fluorescent molecules are removed and the MTs are fixed by perfusion. The MTs can ...
[摘要] [摘要]微管（MT）是细胞骨架中最刚性的组成部分。然而，它们在细胞环境中经常显得高度弯曲，并且控制它们整体形状的机理了解甚少。当前，体外微管分析主要依靠电子显微镜进行高分辨率分析，而全内反射荧光（TIRF ）显微镜则可以对活的荧光标记的微管和相关蛋白进行成像。为了对具有微米曲率的微管进行三维分析，我们开发了一种在体外聚合MT的检测方法 用类似于常规TIRF显微镜操作规程的方式将MT种子的MT粘附到载玻片上。除去游离的荧光分子，并通过灌注固定MTs。然后可以使用带有Airyscan模块的共聚焦显微镜观察MT，以获得更高的分辨率。该协议允许对保留其原始三维形状并与高分辨率免疫荧光检测兼容的微管进行成像。
[背景]微管（MT）是通过异源二聚体的组合制成的聚合物α和β微管蛋白，并且是细胞骨架的主要成分。他们参与了细胞功能的基本机制，如有丝分裂，细胞内转运，胞质分裂和细胞形态的维持（Akhmanova和Steinmetz，2015）。尽管MT本身具有很高的刚性，但它们通常会在细胞中弯曲并产生一些蛋白，从而弯曲微管（Brangwynne等人，2006; Bechstedt等人，2014; Leung等人，2020; Cuveillier等人，2020 ...
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| 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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