Keith C H, Blane K
Department of Zoology, University of Georgia, Athens 30602.
J Neurochem. 1990 Apr;54(4):1258-68. doi: 10.1111/j.1471-4159.1990.tb01957.x.
The site at which tubulin enters into polymer in the neuritic process is a very important datum in terms of our understanding of the mechanism of transport of the microtubular cytoskeleton out the axon. If the form of tubulin being transported out the axon is the microtubule, then assembly of tubulin into microtubules should occur at or near the cell body; if, however, the form of tubulin transported is free tubulin dimer, then assembly can occur at any free microtubule end out the neurite. We have injected a fluorescent analog of tubulin into differentiated PC 12 cells and used differential extraction protocols to extract free dimer but not microtubules. We have imaged these cells before and after extraction by low-light-level video fluorescence microscopy and have used image analysis to examine the sites of tubulin incorporation into polymer or other unextracted components as a function of time. We find that tubulin in the distal reaches of the neurite is found initially as monomer and that its appearance in the unextracted component occurs later. This pattern of appearance of fluorescent tubulin initially in the soluble fraction and later in the unextractable component is qualitatively similar to that reported by other workers for biotinylated tubulin, but we see a larger gap between the rates of appearance in soluble fraction and in polymer. Quantitative analysis of fluorescence intensities in the two compartments with distance out the neurite reveals substantial variation between different neurites: In some neurites, the pattern of variation of unextracted/total tubulin suggests that tubulin enters into the unextracted component primarily near the cell body and that this unextracted component moves out the neurite with time, and in other neurites it suggest that monomer adds into microtubule ends staggered out the neurite. In no case do we see a pattern suggesting that distal addition predominates. These analyses of fluorescence intensities in extracted and unextracted neurites suggest that both transport of polymerized microtubules and monomer addition onto staggered microtubule ends occur in PC12 neurites and that in individual neurites one or the other of these two behaviors may predominate.
就我们对微管细胞骨架向轴突外运输机制的理解而言，微管蛋白在神经突过程中聚合成聚合物的位点是一个非常重要的数据。如果向轴突外运输的微管蛋白形式是微管，那么微管蛋白组装成微管应该发生在细胞体或其附近；然而，如果运输的微管蛋白形式是游离的微管蛋白二聚体，那么组装可以发生在神经突外任何游离的微管末端。我们已将一种微管蛋白的荧光类似物注入分化的PC12细胞，并使用差异提取方案来提取游离二聚体而非微管。我们通过低光水平视频荧光显微镜对提取前后的这些细胞进行了成像，并使用图像分析来检查微管蛋白掺入聚合物或其他未提取成分的位点随时间的变化。我们发现，神经突远端的微管蛋白最初以单体形式存在，其在未提取成分中的出现较晚。荧光微管蛋白最初出现在可溶部分，随后出现在不可提取成分中的这种模式，在质量上与其他研究人员报道的生物素化微管蛋白的模式相似，但我们看到其在可溶部分和聚合物中的出现速率之间的差距更大。对两个区室中荧光强度随神经突长度的定量分析显示，不同神经突之间存在显著差异：在一些神经突中，未提取/总微管蛋白的变化模式表明，微管蛋白主要在细胞体附近进入未提取成分，并且这种未提取成分随时间向神经突外移动，而在其他神经突中则表明单体添加到沿神经突交错排列的微管末端。在任何情况下，我们都没有看到表明远端添加占主导的模式。对提取和未提取神经突中荧光强度的这些分析表明，聚合微管的运输和单体添加到交错排列的微管末端这两种情况在PC12神经突中都存在，并且在单个神经突中，这两种行为中的一种或另一种可能占主导。
