Imaging Diversity in Slow Axonal Transport.
Imaging Diversity in Slow Axonal Transport.
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慢速轴突运输的成像多样性。
DOI：
10.1007/978-1-0716-1990-2_8
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Roy,Subhojit
中科院分区：
文献类型：
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作者：
Ganguly,Archan;Roy,Subhojit
神经元的极化形态需要将索马中合成的蛋白质沿着轴突长度递送至远端突触;这对于维持神经元之间的通信至关重要。这种蛋白质沿沿着轴突转运的组成性和动态过程称为“轴突转运”，其最初特征在于经典的脉冲追踪放射性标记研究，该研究确定了两个主要的速率组分：快组分和慢组分。早期的放射性标记研究表明，缓慢运输货物的“内聚共运输”。然而，这种方法不能用于可视化或提供对这个高度动态过程的机械见解。荧光和光活化成像探针的出现现在已经能够对轴突运输进行实时成像。传统的荧光探针有助于可视化和表征囊泡蛋白运输的分子机制。这些蛋白质通常在轴突运输的快速组分中移动，并沿沿着呈现为“点状结构”。然而，绝大多数在运输的缓慢组分中移动的运输蛋白质，当标记到常规荧光探针时，通常沿着轴突显示“均匀的扩散发光”。这使得在真实的时间内明确地跟踪它们变得具有挑战性。我们的实验室已经使用可光活化的荧光探针来标记在轴突运输的缓慢组分中移动的三种单独的胞质蛋白，并确定了沿着轴突的三种不同的运输模式。我们从这些实验中获得的数据与基于经典放射性标记研究的流行假设相矛盾，该假设认为所有慢转运蛋白都可能作为一个大的大分子蛋白质复合物沿着轴突移动。虽然其他实验室已经开始使用光活化来研究胞质蛋白的轴突运输，但这种技术在很大程度上仍然没有得到充分利用。在这里，我们描述了详细的协议，图像和分析轴突运输的三个典型的慢成分货物沿着轴突培养的海马神经元。
The polarized morphology of neurons necessitates the delivery of proteins synthesized in the soma along the length of the axon to distal synapses; critical for sustaining communication between neurons. This constitutive and dynamic process of protein transport along axons termed “axonal transport” was initially characterized by classic pulse-chase radiolabeling studies which identified two major rate components: a fast component and a slow component. Early radiolabeling studies indicated “cohesive co-transport” of slow transport cargos. However, this approach could not be used to visualize or provide mechanistic insights on this highly dynamic process. The advent of fluorescent and photoactivatable imaging probes have now enabled real-time imaging of axonal transport. Conventional fluorescent probes have helped visualize and characterize the molecular mechanisms of transport of vesicular proteins. These proteins typically move in the fast component of axonal transport and appear as “punctate structures” along axons. However, a large majority of transported proteins that move in the slow component of transport, typically show a “uniform diffusive glow” along axons when tagged to conventional fluorescent probes. This makes it challenging to unequivocally track them in real time. Our lab has used photoactivatable fluorescent probes to tag three individual cytosolic proteins moving in the slow component of axonal transport, and identified three distinct modes of transport along axons. Our data from these experiments argue against the prevailing hypothesis based on classic radiolabeling studies, which suggested that all slow-transport proteins may move along the axon as one large macromolecular protein complex. Although other labs have started using photoactivation to study axonal transport of cytosolic proteins, this technique remains largely under-utilized. Here, we describe the detailed protocols to image and analyze axonal transport of three typical slow-component cargoes along axons of cultured hippocampal neurons.