Elucidation of molecular signaling mechanism of axonal guidance

坍缩反应中介蛋白（CRMPs）与轴突引导信号传导有关，包括信号蛋白。我之前已经发现了一种新的crmp相关蛋白，命名为CRAM (CRMP-associated Molecule)，属于unc-33基因家族。该基因编码563个氨基酸，与二氢嘧啶酶（DHPase）的同源性为57%，与CRMPs的同源性为50- 51%。系统发育树分析表明，CRAM序列与DHPase的相似性最大，与4种crmp的差异最大，表明CRAM与DHPase的亲缘关系比与4种crmp的亲缘关系更密切。CRAM在大脑中表达显著，在胎儿和新生儿中表达特别高，而在成人大脑中表达极低。因此，CRAM是该基因家族的一个独特的新成员，可能在轴突引导信号中发挥作用，不同于其他四种crmp。此外，我最近报道了CRAM与几种细胞蛋白相关，包括酪氨酸激酶Fes/Fps和线粒体septin。在这项研究中，我研究了CRAM在发育神经元中的分布和功能。免疫组化分析显示生长锥丝状伪足有CRAMin积累。细胞松弛素D的实验表明，CRAM的丝状定位与丝状肌动蛋白无关。CRAM在神经元细胞中的过表达可显著促进丝足的生长，导致生长锥体的额外形成，从而获得对信号蛋白3a刺激的抗性。最后，通过RNA干扰敲除CRAM阻断了丝状面形成，揭示了生长锥的异常形态。笔者认为，CRAM调节了丝线动力学和生长锥的发育，从而限制了生长锥对排斥引导信号的反应。

Collapsin response mediator proteins (CRMPs) have been implicated in signaling of axonal guidance, including semaphorins. I have previously identified a novel CRMP-associated protein, designated CRAM for CRMP-Associated Molecule that belongs to the unc-33 gene family. The deduced amino acid sequence reveals that the CRAM gene encodes a protein of 563 amino acids and shows 57 % identity with dihydropyrimidinase (DHPase) and 50-51 % identity with CRMPs. A phylogenetic tree analysis indicates that CRAM sequence shows the greatest similarity with DHPase and divergence from the four CRMPs, indicating that CRAM is more closely related to DHPase than to the four CRMPs. The expression of CRAM is remarkable for brain, and especially high in fetal and neonatal, but decreases to very low levels in adult brain. Thus, CRAM is a unique new member of this gene family and may play a role in axonal guidance signaling distinct from other four CRMPs. Furthermore I have recently reported that CRAM was associated with several cellular proteins including tyrosine kinase Fes/Fps and mitochondrial septin.In this study, I have examined the distribution and function of CRAM in developing neurons. Immunohistochemical analysis showed accumulation of CRAMin the filopodia of growth cones. Experiments using cytochalasin D indicated that filopodial localization of CRAM was independent of filamentous actin. Overexpression of CRAM in neuronal cells significantly promoted filopodial growth and led to the formation of supernumerary growth cones, which acquired resistance to semaphorin-3A stimulation. Finally, knockdown of CRAM by using RNA interference blocked filopodial formation and revealed an aberrant morphology of growth cones. I propose that CRAM regulates filopodial dynamics and growth cone development, thereby restricting the response of growth cone to repulsive guidance cues.