Our understanding of endocytic pathway dynamics is restricted by the diffraction limit of light microscopy. Although super-resolution techniques can overcome this issue, highly crowded cellular environments, such as nerve terminals, can also dramatically limit the tracking of multiple endocytic vesicles such as synaptic vesicles (SVs), which in turn restricts the analytical dissection of their discrete diffusional and transport states. We recently introduced a pulse-chase technique for subdiffractional tracking of internalized molecules (sdTIM) that allows the visualization of fluorescently tagged molecules trapped in individual signaling endosomes and SVs in presynapses or axons with 30- to 50-nm localization precision. We originally developed this approach for tracking single molecules of botulinum neurotoxin type A, which undergoes activity-dependent internalization and retrograde transport in autophagosomes. This method was then adapted to localize the signaling endosomes containing cholera toxin subunit-B that undergo retrograde transport in axons and to track SVs in the crowded environment of hippocampal presynapses. We describe (i) the construction of a custom-made microfluidic device that enables control over neuronal orientation; (ii) the 3D printing of a perfusion system for sdTIM experiments performed on glass-bottom dishes; (iii) the dissection, culturing and transfection of hippocampal neurons in microfluidic devices; and (iv) guidance on how to perform the pulse-chase experiments and data analysis. In addition, we describe the use of single-molecule-tracking analytical tools to reveal the average and the heterogeneous single-molecule mobility behaviors. We also discuss alternative reagents and equipment that can, in principle, be used for sdTIM experiments and describe how to adapt sdTIM to image nanocluster formation and/or tubulation in early endosomes during sorting events. The procedures described in this protocol take ∼1 week.

中文翻译：

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通过内在分子的亚微分跟踪可视化活神经元中的内吞再循环和运输。

我们对内吞途径动力学的理解受到光学显微镜的衍射极限的限制。尽管超分辨率技术可以解决此问题，但高度拥挤的细胞环境（例如神经末梢）也可以极大地限制对多个内吞囊泡（例如突触囊泡（SVs））的跟踪，从而限制了对它们离散扩散和扩散的分析解剖。运输状态。我们最近推出了一种脉冲追逐技术，用于对内部化分子（sdTIM）进行超微分跟踪，该技术可使可视化的荧光标记分子以30至50 nm的定位精度可视化捕获在突触前突突或轴突中的单个信号内体和SV中。我们最初开发这种方法来跟踪A型肉毒杆菌神经毒素的单分子，在自噬体中进行依赖于活性的内在化和逆行转运。然后，该方法适用于定位含有霍乱毒素亚基B的信号传递内体，该内体在轴突中进行逆行运输，并在海马突触前拥挤的环境中追踪SV。我们描述（i）能够控制神经元方向的定制微流控设备的构造；（ii）在玻璃底皿上进行sdTIM实验的灌注系统的3D打印；（iii）在微流控装置中解剖，培养和转染海马神经元；（iv）有关如何进行脉冲追踪实验和数据分析的指南。此外，我们描述了使用单分子跟踪分析工具来揭示平均和异构单分子流动性行为。我们还将讨论原则上可用于sdTIM实验的替代试剂和设备，并描述如何在分类事件期间使sdTIM适应早期内体中纳米簇的形成和/或成管。该协议中描述的过程大约需要1周。