Bernstein B W, Bamburg J R
Department of Biochemistry, Colorado State University, Fort Collins 80523.
Mol Neurobiol. 1992 Summer-Fall;6(2-3):95-106. doi: 10.1007/BF02780546.
Does actin in the emerging axons of regenerating neurons arise from the assembled or unassembled actin pool in the cell soma? We investigated this question by loading neurons with one of two fluorescently labeled molecules: rhodamine actin (r-actin) and rhodamine phalloidin (r-phalloidin). The assembly behavior of r-actin in vitro was identical to unlabeled actin. R-phalloidin binds tightly only to the filamentous form of actin (F-actin) and stabilizes filaments against disassembly. Hence, r-phalloidin-tagged filaments should be less likely to disassemble than r-actin-tagged filaments. Neurons of 10-d-old chick embryos were loaded with r-actin or r-phalloidin by triturating trypsinized dorsal root ganglia in isotonic sucrose containing the fluorescently tagged molecule. Isolated neurons were plated on glass coverslips in modified L15 medium containing nerve growth factor. Video images of the live cells on a thermoregulated stage were acquired with a computer imaging system. After 24 h in culture, the fluorescence distribution of r-phalloidin and r-actin was examined in live neurons of comparable morphology, neurite outgrowth, and intensity of somal fluorescence. Greater than 90% of the neurons labeled with r-actin (n = 81) contained detectable levels of fluorescence in emerging neurite fibers, often extending to the tip of the growing process. Less than 10% of the neurons labeled with r-phalloidin (n = 53) contained any fluorescence in the neurite fibers. In those that did contain fluorescence, the r-phalloidin usually was confined to the proximal segment of the neurite, and in no case was it found at the growing tip. Confocal microscopy and cooled CCD imaging of fixed neurons showed that all structures that incorporated r-actin or r-phalloidin also stained with bodipy phallacidin. This colocalization confirms the association of rhodamine-tagged species with F-actin. Our data support a model in which actin, needed in early stages of neurite outgrowth, arises from a pool in the soma that is capable of disassembly.
再生神经元新生轴突中的肌动蛋白是源自细胞体中已组装的肌动蛋白池还是未组装的肌动蛋白池呢？我们通过用两种荧光标记分子之一加载神经元来研究这个问题：罗丹明肌动蛋白（r-肌动蛋白）和罗丹明鬼笔环肽（r-鬼笔环肽）。r-肌动蛋白在体外的组装行为与未标记的肌动蛋白相同。r-鬼笔环肽仅紧密结合肌动蛋白的丝状形式（F-肌动蛋白），并稳定细丝以防解聚。因此，r-鬼笔环肽标记的细丝比r-肌动蛋白标记的细丝更不容易解聚。通过在含有荧光标记分子的等渗蔗糖中研磨胰蛋白酶处理的背根神经节，将10日龄鸡胚的神经元用r-肌动蛋白或r-鬼笔环肽加载。将分离的神经元接种在含有神经生长因子的改良L15培养基中的玻璃盖玻片上。使用计算机成像系统获取在温度调节台上的活细胞的视频图像。培养24小时后，在形态、神经突生长和胞体荧光强度相当的活神经元中检查r-鬼笔环肽和r-肌动蛋白的荧光分布。用r-肌动蛋白标记的神经元中超过90%（n = 81）在新生神经突纤维中含有可检测水平的荧光，这些荧光通常延伸到生长过程的尖端。用r-鬼笔环肽标记的神经元中不到10%（n = 53）在神经突纤维中含有任何荧光。在那些确实含有荧光的神经元中，r-鬼笔环肽通常局限于神经突的近端部分，并且在生长尖端从未发现过。固定神经元的共聚焦显微镜和冷却电荷耦合器件成像显示，所有结合r-肌动蛋白或r-鬼笔环肽的结构也用硼二吡咯亚甲基鬼笔环肽染色。这种共定位证实了罗丹明标记的物质与F-肌动蛋白的关联。我们的数据支持一种模型，即神经突生长早期所需的肌动蛋白源自细胞体中一个能够解聚的池。
