Resolving the In Situ Three-Dimensional Structure of Fly Mechanosensory Organelles Using Serial Section Electron Tomography

Landi Sun, Jana Meissner, Jianfeng He, Lihong Cui, Tobias Fürstenhaupt, Xin Liang
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Abstract

Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples. Key features • Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET). • Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms. • The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details.
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利用序列切片电子断层扫描解析蝇类机械感觉细胞器的原位三维结构
机械感觉细胞器(MO)是一种特化的亚细胞实体,其中的力敏通道和支持结构(如微管细胞骨架)有序地组织在一起。MOs的微妙结构亟待解决,以了解它们检测力的机制以及它们是如何形成的。在这里,我们介绍了一种利用序列切片电子断层扫描(SS-ET)获取蝇类MO纳米超微结构详细信息的方法。为了保留细微的结构细节,首先使用高压冷冻机对组织进行低温固定,然后在低温下进行冷冻置换,并在室温下嵌入树脂中。然后,制备样本切片,用于获取双轴倾斜系列图像,并进一步处理以进行断层重建。最后,利用微管作为靶标,将连续切片的断层图像合并成一个较大的体积。利用这一方案,我们成功地重建了感觉器,为了解蝇类机械感觉器如何工作和形成提供了新的分子见解。根据我们的经验,我们认为只需稍加修改,该方案就能适用于使用不同细胞和组织样本的广泛应用。主要特点 - 利用序列切片电子断层扫描(SS-ET)解析亚细胞器的高分辨率三维超微结构。- 与单轴倾斜系列相比,双轴倾斜系列的傅立叶空间覆盖范围更广,从而提高了重建断层图的分辨率和特征。- 高压冷冻和冷冻置换的使用最大限度地保留了精细结构细节。
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