Sun Landi, Meissner Jana, He Jianfeng, Cui Lihong, Fürstenhaupt Tobias, Liang Xin
IDG/McGovern Institute for Brain Research, School of Life Sciences, Tsinghua University, Beijing, China.
Guangzhou National Laboratory, Guangzhou, China.
Bio Protoc. 2024 Feb 20;14(4):e4940. doi: 10.21769/BioProtoc.4940.
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.
机械感觉细胞器（MOs）是特殊的亚细胞实体，力敏通道和支撑结构（如微管细胞骨架）在其中有序排列。MOs的精细结构需要解析，以了解它们检测力的机制以及它们是如何形成的。在这里，我们描述了一种方案，该方案允许通过使用连续切片电子断层扫描（SS-ET）获得关于果蝇MOs纳米级超微结构的详细信息。为了保留精细的结构细节，使用高压冷冻机对组织进行冷冻固定，随后在低温下进行冷冻置换，并在室温下包埋在树脂中。然后，制备样品切片并用于获取双轴倾斜系列图像，对其进行进一步处理以进行断层重建。最后，使用微管作为基准标记，将连续切片的断层图组合成一个更大的单一体积。使用该方案，我们成功重建了感觉细胞器，这为果蝇机械感觉细胞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，只需进行最小的修改，该方案就可以适用于使用不同细胞和组织样本的广泛应用。关键特性 • 使用连续切片电子断层扫描（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，双轴倾斜系列在傅里叶空间提供了更广泛的覆盖范围，提高了重建断层图的分辨率和特征。 • 使用高压冷冻和冷冻置换最大程度地保留了精细的结构细节。
