Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
Structure. 2009 Nov 11;17(11):1433-41. doi: 10.1016/j.str.2009.09.008.
Microtubules are essential cytoskeletal tracks for cargo transportation in axons and also serve as the primary structural scaffold of neurons. Structural assembly, stability, and dynamics of axonal microtubules are of great interest for understanding neuronal functions and pathologies. However, microtubules are so densely packed in axons that their separations are well below the diffraction limit of light, which precludes using optical microscopy for live-cell studies. Here, we present a single-molecule imaging method capable of resolving individual microtubules in live axons. In our method, unlabeled microtubules are revealed by following individual axonal cargos that travel along them. We resolved more than six microtubules in a 1 microm diameter axon by real-time tracking of endosomes containing quantum dots. Our live-cell study also provided direct evidence that endosomes switch between microtubules while traveling along axons, which has been proposed to be the primary means for axonal cargos to effectively navigate through the crowded axoplasmic environment.
微管是轴突中货物运输的重要细胞骨架轨道，也是神经元的主要结构支架。了解神经元的功能和病理，需要研究轴突微管的结构组装、稳定性和动力学。然而，微管在轴突中排列得非常紧密，以至于它们之间的分离度远低于光的衍射极限，这使得光学显微镜无法用于活细胞研究。在这里，我们提出了一种能够在活轴突中分辨单个微管的单分子成像方法。在我们的方法中，通过跟踪沿着微管行进的单个轴突货物来揭示未标记的微管。我们通过实时追踪含有量子点的内体，在 1 微米直径的轴突中分辨出了超过 6 个微管。我们的活细胞研究还提供了直接证据，证明内体在沿着轴突运动时在微管之间切换，这被认为是轴突货物有效穿越拥挤的轴浆环境的主要手段。
