ANALYSIS OF MOLECULAR MECHANISMS FOR NEURONAL POLARITY AND AXON FORMATION BY CRMP-2

发现CRMP-2参与神经元极性和轴突的形成轴突和树突的形成是神经元发育的基本步骤。在本研究中，我们发现CRMP-2在培养海马神经元的生长轴突中富集。细胞中过量表达CRMP-2导致多余轴突的形成，但神经突总数未显著增加。CRMP-2不仅在轴突形成初期促进了多个轴突的形成，而且在已经形成的树突上诱导轴突萌发。此外，截断的CRMP-2突变体的表达以显性负向方式抑制初级轴突的形成。因此，神经突中局部浓度的CRMP-2似乎在海马神经元轴突诱导中起关键作用，从而建立和维持神经元极性。为了进一步研究CRMP-2诱导轴突形成的分子机制，我们还研究了CRMP-2的相关蛋白。在这里，我们确定了CRMP-2的两种活性：微管蛋白-异二聚体结合和促进微管组装。CRMP-2结合的小管二聚体比其结合的微管具有更高的亲和力。在CRMP-2存在的情况下，微管聚合增强了CRMP-2与微管的结合。CRMP-2与微管蛋白的结合特性与优先结合微管的Tau蛋白明显不同。在神经元中，过表达CRMP-2促进轴突生长和分支。CRMP-2突变体缺乏负责微管组装的区域，以显性负向方式抑制轴突生长和分支。综上所述，我们的研究结果表明，CRMP-2作为小管蛋白异二聚体的伙伴，以一种不同于传统map的方式调节轴突生长和分支。通过蛋白质组学策略寻找参与神经元极性和轴突形成的分子我们进一步开始研究除了CRMP-2外，参与轴突和极性形成的分子。为了寻找参与轴突和极性形成的分子，我们建立了一个高分辨率的2-DE二维凝胶电泳（2-DE）系统。我们使用了14个大的2-DE凝胶（12个24厘米× 70厘米的凝胶和2个18厘米× 70厘米的凝胶），它们被组装成一个93厘米× 103厘米的赛博凝胶。我们的数据表明，网络凝胶可以显示细胞中在1-10^5动态范围内表达的超过11,000个蛋白质点。与标准程序相比，当前策略的使用导致检测到的斑点数量增加了约500%。利用这个系统，我们目前正在筛选在神经元极性形成过程中表达的分子。少

Identification of CRMP-2 as a molecule involved in neuronal polarity and axon formationFormation of an axon and dendrites is a fundamental step in neuronal development. In this research, we found that CRMP-2 was enriched in growing axons of cultured hippocampal neurons. Overexpression of CRMP-2 in the cells led to the formation of supernumerary axons without remarkable increase in the total number of neurites. CRMP-2 not only enhanced the formation of multiple axons during the initial stages of axon specification but also induced axon sprouting from already formed dendrites. Furthermore, expression of truncated CRMP-2 mutants suppressed the formation of primary axon in a dominant negative manner. Thus, local concentrations of CRMP-2 in neurites appear to play a critical role in axon induction of hippocampal neurons, thereby establishing and maintaining neuronal polarity.In order to examine the molecular mechanisms for CRMP-2 induced axonal formation, we also investigated the CRMP-2 bin … More ding proteins. Here, we identified two activities of CRMP-2: tublin-heterodimer binding and the promotion of microtuble assembly. CRMP-2 bound tublin dimers with higher affinity than it bound microtubules. Association of CRMP-2 with microtubules was enhanced by tublin polymerization in the presence of CRMP-2. The binding property of CRMP-2 with tubulin was apparently distinct from that of Tau, which preferentially bound microtubules. In neurons, overexpression of CRMP-2 promoted axonal growth and branching. A mutant of CRMP-2, lacking the region responsible for microtuble assembly, inhibited axonal growth and branching in a dominant-negative manner. Taken together, our results suggest that CRMP-2 regulate axonal growth and branching as a partner of the tublin hetereodimer, in a different fashion from traditional MAPs.Search for molecules involved in neuronal polarity and axon formation by protemic strategyWe further started to investigated the molecules involved in axon and polarity formation in addition to CRMP-2 by proteomic strategy. In order to searched molecules involved in axon and polarity formation, we established a 2-DE two-dimensional gel electrophoresis (2-DE) system of highresolution. We utilized fourteen large 2-DE gels (twelve 24 cm x 70 cm gels and two 18 cm x 70 cm gels) which were assembled into a 93 cm x 103 cm cybergel. Our data suggested that the cyber gel can display more than 11,000 protein spots expressed in a 1-10^5 dynamic range in cells. The utilization of present strategy led to about 500% increase in the number of detected spots in comparison to a standard procedure. With this system, we are currently screening molecules which are expressed during the processes of neuronal polarity formation. Less