| 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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| Microtubule Seeded-assembly in the Presence of Poorly Nucleating Nucleotide Analogues
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Author:
Date:
2020-08-20
[Abstract] Microtubule dynamic instability is driven by the hydrolysis of the GTP bound to the β-subunit of the α-β tubulin heterodimer. Nucleotide analogues are commonly used to mimic the different steps of the tubulin GTPase cycle, but most of them are poor microtubule nucleators. Usually, microtubule assembly is seeded by guanylyl-(α, β)-methylene-diphosphonate (GMPCPP) or glycerol that can be limiting factors in monitoring the effect of other nucleotide analogs on their polymerization. Here, we describe a protocol that allows the assembly of microtubules in the presence of nucleotide analogues without the need of heterogeneous seeds and at a low final glycerol concentration. Microtubules are first assembled in the presence of the analogue of interest and glycerol to promote assembly. These ...
[摘要] [摘要 ] 微管动态不稳定性是由与α - β 微管蛋白异二聚体的β- 亚基结合的GTP水解驱动的。核苷酸类似物通常用于模拟微管蛋白GTPase循环的不同步骤，但其中大多数是不良的微管成核剂。通常，微管组装是通过胍基-（α ，β ）-亚甲基-二膦酸酯（GMPCPP）或甘油来接种的，它们可能是限制其他核苷酸类似物对其聚合作用的监测因素。 在这里，我们描述了一种协议，该协议允许在核苷酸类似物存在的情况下组装微管，而无需异质种子，并且最终甘油浓度低。首先在感兴趣的类似物和甘油的存在下组装微管以促进组装。然后对这些微管进行超声处理，以产生种子，这些种子将在不存在甘油的情况下用于组装微管。这种策略产生了均质的核苷酸结合的微管，可以通过生化或结构方法（如冷冻电子显微镜）进一步分析。
[背景 ] 核苷酸类似物通常用于研究的构象变化，该α - β 微管组装和水解绑定到其可交换的（E）的网站上的GTP的期间微管蛋白异源二聚体经历。但是，除GMPCPP以外，大多数类似物都是微管成核剂。为克服这一困难，GMPCPP稳定化的种子通常用于存在其他类似物的情况下延长微管（Maurer 等人，2011和2014； Zhang ...
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| Preparing Immunolocalization Slides of Maize Meiotic Chromosomes for Three-dimensional Microscopy
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Author:
Date:
2020-07-20
[Abstract] The protocol provides fully detailed steps for preparing microscopic slides of acrylamide-embedded maize meiotic cells. This method is particularly useful for examining chromatin structure and chromosome arrangement without destroying the three-dimensional organization of the nucleus.
[摘要] [摘要] 该协议提供了制备丙烯酰胺包埋的玉米减数分裂细胞显微玻片的完整详细步骤。该方法对于检查染色质结构和染色体排列而不破坏细胞核的三维结构特别有用。
[背景] 减数分裂是一个动态过程，涉及同源染色体配对，突触和重组。用于研究减数分裂蛋白的定位和动力学的细胞学分析对于了解这些过程的细节至关重要。在许多显微载玻片制备方法中，空间染色质的组织受到机械加工或化学溶剂的破坏。在这里，我们描述了三维显微镜协议，以分析染色质结构和减数分裂蛋白的定位，而不会干扰核组织。
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