HOW DO NEURONS REPLACE VIMENTIN WITH NEUROFILAMENTS?

可塑性神经突向稳定轴突的转变是最重要的 突触正常发育和维持的重要性 连接. 神经元中间体的分级表达 纤维（IF）伴随着这些事件，其中神经元依赖于 通用波形蛋白IF系统启动高塑性的产物， 假定的轴突神经突，然后随后存款神经丝（NF） 在突触形成过程中稳定这些结构， 神经突触 虽然很明显Vm的表达 在此期间，神经元的数量下降，神经纤维的数量增加， 在存在现有Vm网络的情况下实现该转换 仍不清楚。 我们的数据表明，虽然大部分Vm和NF形成离散IF， 网络，一部分新表达的NF亚基（特别是NF-H） 在积累之前， 神经纤维 这些发现表明，Vm细丝的更快周转 与NF相反，可能有助于发展过渡， 这些网络。 本试点方案的研究将调查（1）相对于 Vm和NF的周转率和（2） VM-NF杂聚体在NB 2a/d1神经母细胞瘤分化中的作用 细胞和鸡背根神经节神经元的培养，使用我们的 显微注射、免疫荧光、共聚焦显微镜 显微镜，免疫电子显微镜，显微密度测定， 放射性标记、免疫沉淀、电泳和放射自显影。 这些分析将证实或否定我们的工作假设：（1） 神经元没有办法调节异或 NF均聚物的积累是由 亚基捕获在相对长寿命的NF内， Vm的发育下降，以及（2）任何此类推定的Vm和NF共同作用。 聚合物代表了短暂的组装中间体， 局限于核周体和轴突丘区域，而不是 进行轴突运输 如果这些假设被证明是有效的，他们将证明 神经元通过这种方法影响发育之间的转换， 在整个生命过程中为成熟的轴突提供支持。 如果他们能证明 假的，这项试验性建议的研究将指明方向， 随后的研究表明， 成熟轴突细胞骨架的发育和建立。

The transition of plastic neurites to stabilized axons is of utmost importance in proper development and maintenance of synaptic connections. An hierarchical expression of neuronal intermediate filaments (IFs) accompanies these events, in which neurons rely on the generic vimentin IF system to initiate the outgrowth of highly-plastic, putative axonal neurites, then subsequently deposit neurofilaments (NFs) to stabilize these structures during synaptogenesis and maintain these synapses throughout life. Although it is clear that expression of Vm declines and that of NFs increases during this period, how the neuron effects this transition in the presence of an existing Vm network remains unclear. Our data indicates that, while the bulk of Vm and NFs form discrete IF networks, a portion of newly-expressed NF subunits (in particular, NF-H) undergoes transient association with Vm prior to its accumulation in NFs. These findings suggest that more rapid turnover of Vm filaments as opposed to NFs may contribute to the developmental transition between these networks. Studies in this pilot proposal will investigate (1) the relative turnover rate of Vm and NFs and (2) the subcellular localization of putative Vm-NF heteropolymers in differentiating NB2a/d1 neuroblastoma cells and chick dorsal root ganglion neurons in culture, using our established techniques of microinjection, immunofluorescence, confocal microscopy, immuno-electron microscopy, microdensitometry, radiolabeling, immunoprecipitation, electrophoresis and autoradiography. These analyses will confirm or deny our working hypotheses that (1) neurons have no means to regulate the formation of hetero- or homopolymers, and the accumulation of NF homopolymers is mediated by subunit trapping within relatively long-lived NFs coupled with a developmental decline in Vm, and (2) any such putative Vm and NF co- polymers represent transient assembly intermediates that are largely confined to the perikaryon and axon hillock region rather than undergoing axonal transport. Should these hypotheses be proven valid, they will demonstrate the method by which neurons effect the developmental switch between the that provides support to mature axons throughout life. Should they prove false, the studies of this pilot proposal will point the way towards subsequent investigations of neurofilament subunit exchange during development and establishment of the mature axonal cytoskeleton.