应用超分辨光电融合成像技术研究线粒体融合与分裂的机制

Mitochondrial are high dynamic organelles that continually undergo fusion and fission to maintain its shape. Mitochondrial dynamics has been linked to multiple mitochondrial functions, including apoptosis, energy metabolism, and signal transduction. Limited by the development of imaging techniques, the detail of mitochondrial fusion and fission are not yet clear. The applicant intends to adopt a new developed imaging technique termed correlative cyro superresolution light and electron microscopy to study the molecular mechanism of mitochondrial fusion and fission. This technique benefit from keeping sample in the near native state, specific labeling and high resolution. By using this technique, mitochondrial fusion and fission events can be captured and the detail structure of mitochondrial can be imaged at nanometer resolution. Proteins involved in fusion and fission can be localized precisely on the structure captured from electron microscopy in situ to reveal the molecular mechanism of mitochondrial fusion and fission.

线粒体是高度动态的细胞器，通过频繁的融合与分裂来保持自身形态，参与细胞凋亡、能量代谢、信号转导等重要的细胞生物学过程。受限于显微成像技术的发展，其调控的细节尚不明确。申请人拟采用新发展的冷冻超分辨光电融合成像技术来研究线粒体融合分裂的分子机制。该技术具有保持样品处于近天然状态、特异性标记、分辨率高、导向定位结构观察等优势，可以真实的捕捉融合分裂时线粒体的原位三维结构，并对参与融合分裂的相关蛋白分子进行纳米分辨率的共定位分析，探索融合分裂过程中蛋白复合体的募集、组装和定位变化对线粒体融合分裂的作用，从而揭示调控线粒体融合分裂的分子机制。

按项目计划，我们首先发展了冷冻超分辨光电融合成像技术，通过高压冷冻制备的冷冻超薄切片样品，是目前保存状态最接近样品原始状态的样品制备方法。我们发现荧光蛋白在冷冻成像条件下能发出更多的光子，从而可以提高定位精度。我们利用这种方法成果的把荧光标记的线粒体外膜蛋白定位在三维冷冻断层电镜成像的线粒体外膜上。利用这项技术，我们正在研究线粒体分裂和融合相关蛋白的定位和相互之间关系，以揭示线粒体融合与分裂的分子机制。