A simple photoactivation and image analysis module for visualizing and analyzing axonal transport with high temporal resolution
A simple photoactivation and image analysis module for visualizing and analyzing axonal transport with high temporal resolution
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DOI：
10.1038/nprot.2011.428
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发表时间：
2012-01-01
期刊：
影响因子：
14.8
通讯作者：
Scott, David A.
中科院分区：
文献类型：
--
作者：
Roy, Subhojit;Yang, Ge;Scott, David A.
我们描述了一种策略，用于分析轴突运输的胞质蛋白（CPCPs）使用光活化GFP-PAGFP-与修改的标准成像组件，可以追溯到传统的落射荧光显微镜。光活化和可视化几乎是同时进行的，从而可以研究具有快速移动的组分的蛋白质。用PAGFP标记的构建体转染培养的海马神经元，轴突内的离散蛋白质群体被光活化，然后通过实时成像跟踪活化的群体。我们展示了这种方法在分析具有固有的扩散池的CPCPs的轴突运输中的实用性，并将这种运输方式与被动扩散和囊泡运输区分开来。还描述了用于量化运动的分析工具。除了制备神经元培养物/转染所需的时间外，实验需要2-3小时，在此期间可以对几个轴突进行成像和分析。这些方法应该很容易被大多数实验室采用，也可能有助于监测CP在其他细胞类型中的运动。
We describe a strategy for analyzing axonal transport of cytosolic proteins (CPCPs) using photoactivatable GFP-PAGFP-with modifications of standard imaging components that can be retroactively fitted to a conventional epifluorescence microscope. The photoactivation and visualization are nearly simultaneous, allowing studies of proteins with rapidly mobile fractions. Cultured hippocampal neurons are transfected with PAGFP-tagged constructs, a discrete protein population within axons is photoactivated, and then the activated population is tracked by live imaging. We show the utility of this method in analyzing axonal transport of CPCPs that have inherent diffusible pools and distinguish this transport modality from passive diffusion and vesicle transport. The analytical tools used to quantify the motion are also described. Aside from the time needed for preparation of neuronal cultures/transfection, the experiment takes 2-3 h, during which time several axons can be imaged and analyzed. These methods should be easy to adopt by most laboratories and may also be useful for monitoring CP movement in other cell types.