Rapid intermittent movement of axonal neurofilaments observed by fluorescence photobleaching
Rapid intermittent movement of axonal neurofilaments observed by fluorescence photobleaching
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DOI：
10.1091/mbc.12.10.3257
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发表时间：
2001-10-01
影响因子：
3.3
通讯作者：
Brown, A
中科院分区：
文献类型：
--
作者：
Wang, L;Brown, A
对培养神经元的轴突神经丝阵列中自然存在的间隙的观察表明，神经丝聚合物沿轴突以快速、间歇性和高度异步的方式移动。相比之下，使用激光光漂白对轴突神经丝的研究尚未检测到运动。在这里，我们描述了一种改进的光漂白策略，它可以直接观察神经丝的运动。表达gfp标记的神经丝蛋白的神经元轴突在常规荧光显微镜汞弧灯的激发下漂白12 ~ 60 s。漂白区域的长度从10到60毫米不等。通过漂白具有相对较少神经丝的细轴突，我们能够将荧光强度降低到足以检测到从周围未漂白区域移动过来的神经丝。短时间间隔的延时成像显示荧光灯丝以高达2.8 μ m/s的峰值速率通过漂白区域的快速，间歇性和高度异步运动。运动动力学与我们之前观察到的神经丝通过自然产生的间隙运动非常相似，这表明运动没有受到光漂白过程的损害。这些结果表明，荧光光漂白可以用于研究细胞骨架的缓慢轴突运输。聚合物，但前提是实验策略的设计能够确保快速的异步运动可以被检测到。这可能解释了以前的光漂白研究未能揭示轴突中神经丝蛋白和其他细胞骨架蛋白的运动。
Observations on naturally occurring gaps in the axonal neurofilament array of cultured neurons have demonstrated that neurofilament polymers move along axons in a rapid, intermittent, and highly asynchronous manner. In contrast, studies on axonal neurofilaments using laser photobleaching have not detected movement. Here, we describe a modified photobleaching strategy that does pern-Lit the direct observation of neurofilament movement. Axons of cultured neurons expressing GFP-tagged neurofilament protein were bleached by excitation with the mercury arc lamp of a conventional epifluorescence microscope for 12-60 s. The length of the bleached region ranged from 10 to 60 mum. By bleaching thin axons, which have relatively few neurofilaments, we were able to reduce the fluorescent intensity enough to allow the detection of neurofilaments that moved in from the surrounding unbleached regions. Time-lapse imaging at short intervals revealed rapid, intermittent, and highly asynchronous movement of fluorescent filaments through the bleached regions at peak rates of up to 2.8 mum/s. The kinetics of movement were very similar to our previous observations on neurofilaments moving through naturally occurring gaps, which indicates that the movement was not impaired by the photobleaching process. These results demonstrate that fluorescence photobleaching can be used to study the slow axonal transport of cytoskeletal. polymers, but only if the experimental strategy is designed to ensure that rapid asynchronous movements can be detected. This may explain the failure of previous photobleaching studies to reveal the movement of neurofilament proteins and other cytoskeletal proteins in axons.