Organelle Transport Of Ion Channels In Excitable Cells

这个项目的目的是研究神经退化的机制。神经损伤后发生的一系列特征性事件，统称为沃勒氏变性，如轴突细胞骨架溶解、髓鞘变性、巨噬细胞侵袭神经和雪旺细胞增殖。这些过程通常在横断后24-48小时完成。在突变小鼠模型C57BL/Ola株中，变性异常延迟。在这些其他方面看起来正常的小鼠中，轴突在横断后2周内仍保持其传导动作电位的能力，并在结构上保持完整长达1个月。我们一直在使用横切的巨型轴突，在体内，从鱿鱼作为模型系统来理解这种延迟版本的沃勒氏变性。这些轴突在横断2天后仍保持其传导动作电位的能力，其细胞骨架也保持完整。然而，我们注意到通过视频增强对比度差干涉对比显微镜（VEC-DIC）测量的快速轴突流量发生了显著变化。控制轴突包含大量主要沿正方向运动的小颗粒，我们之前已经证明了这些小颗粒是直径30纳米的运输囊泡。一小部分囊泡含有轴突电压门控钾通道。其他囊泡可能含有其他蛋白质货物，也运往腋膜。所有的囊泡都含有一个拷贝的基于微管的运动蛋白，运动蛋白，以及一个拷贝的基于肌动蛋白的运动蛋白，肌球蛋白- ii。后者的唯一位点似乎是这些运输囊泡。横断轴突的vecc - dic记录显示，正方向的交通几乎完全丧失，逆行方向的交通很少或没有改变。此外，使用免疫技术（免疫印迹和免疫细胞化学）在横切轴突的轴质中未观察到肌球蛋白- ii和钾通道。我们已经得出结论，运输囊泡的丢失似乎是延迟性沃勒氏变性的第一步。

The purpose of this project is to investigate the mechanisms which underlie nerve degeneration. A characteristic sequence of events occur following nerve injury, collectively termed Wallerian degeneration, such as dissolution of the axonal cytoskeleton, degeneration of the myelin sheath, invasion of the nerve by macrophages, and Schwann cell proliferation. These processes are usually complete 24-48 hr following transection. Degeneration is extraordinarily delayed in a mutant mouse model, the C57BL/Ola strain. In these otherwise normal appearing mice, axons retain their ability to conduct action potentials up to 2 weeks after transection, and they remain structurally intact for as long as 1 month. We have been using transected giant axons, in vivo, from squid as a model system to understand this delayed version of Wallerian degeneration. These axons retain their ability to conduct action potentials 2 days following transection, and their cytoskeleton also remains intact. However, we have noticed striking changes in fast axonal traffic measured with video enhanced contrast, differential interference contrast microscopy (VEC-DIC). Control axons contain a large number of small particles moving primarily in the orthograde direction, which we have previously demonstrated are transport vesicles 30 nm in diameter. A small fraction of the vesicles contain the axonal, voltage-gated potassium channel. The other vesicles presumably contain other protein cargo also destined for the axolemma. All of the vesicles contain one copy of the microtubule based motor protein, kinesin, as well as one copy of the actin based motor protein, myosin-II. The sole locus of the latter appears to be these transport vesicles. VEC-DIC recordings from transected axons reveal an almost complete loss of orthograde traffic, with little or no alteration of traffic in the retrograde direction. Moreover, myosin-II and potassium channels were not observed in the axoplasm of transected axons using immunological techniques (immunoblots and immunocytochemistry). We have concluded that the loss of transport vesicles appears to be the first step underlying delayed Wallerian degeneration.