Gabriel Luke R, Wu Sijia, Melikian Haley E
Program in Neuroscience, Graduate School of Biomedical Sciences, University of Massachusetts Medical School.
Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, University of Massachusetts Medical School;
J Vis Exp. 2014 Apr 3(86):51240. doi: 10.3791/51240.
Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale. There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation. However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons. Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms. Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation. We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons. This approach is likely to have broad utility in the field of neuronal endocytic trafficking.
受调控的内吞运输是促进各种神经调节事件的核心机制，它通过在分钟时间尺度上动态控制受体、离子通道和转运蛋白在细胞表面的呈现来实现。控制单个蛋白质内吞运输的机制多种多样。研究运输分子基础的研究主要依赖表面生物素化来定量测量膜蛋白表面表达对外源刺激和基因操作的变化。然而，这种方法主要局限于培养细胞，这可能无法如实反映成年神经元中起作用的生理相关机制。此外，培养细胞方法可能会低估运输机制中的区域特异性差异。在这里，我们描述了一种将细胞表面生物素化扩展到急性脑片制备的方法。我们证明这种方法提供了一种高保真方法来测量成年神经元中膜蛋白表面水平的快速变化。这种方法可能在神经元内吞运输领域具有广泛的用途。
