RESULTS: We examined the contributions of Rasal1 to neuronal development in primary culture of hippocampal neurons through modulation of Rasal1 expression using molecular tools. Fixed and live cell imaging demonstrate diffuse expression of Rasal1 throughout the cell soma, dendrites and axon which localizes to the neuronal plasma membrane in response to intracellular calcium fluctuation. Pull-down and co-immunoprecipitation demonstrate direct interaction of Rasal1 with PKC, tubulin, and CaMKII. Consequently, Rasal1 is found to stabilize microtubules, through post-translational modification of tubulin, and accordingly inhibit dendritic outgrowth and branching. Through imaging, molecular, and electrophysiological techniques Rasal1 is shown to promote NMDA-mediated synaptic activity and CaMKII phosphorylation.
CONCLUSIONS: Rasal1 functions in two separate roles in neuronal development; calcium regulated neurite outgrowth and the promotion of NMDA receptor-mediated postsynaptic events which may be mediated both by interaction with direct binding partners or calcium-dependent regulation of down-stream pathways. Importantly, the outlined molecular mechanisms of Rasal1 may contribute notably to normal neuronal development and synapse formation.
结果:我们使用分子工具通过调节Rasal1表达,研究了Rasal1对海马神经元原代培养物中神经元发育的贡献。固定和活细胞成像显示Rasal1在整个细胞瘤中弥漫性表达,树突和轴突定位于神经元质膜,以响应细胞内钙波动。下拉和免疫共沉淀表明Rasal1与PKC直接相互作用,微管蛋白,还有CaMKII.因此,发现Rasal1可以稳定微管,通过微管蛋白的翻译后修饰,并相应地抑制树突状生长和分支。通过成像,分子,和电生理技术Rasal1显示可促进NMDA介导的突触活性和CaMKII磷酸化。
结论:Rasal1在神经元发育中起两种不同的作用;钙调节神经突生长和促进NMDA受体介导的突触后事件,这可能是通过与直接结合配偶体的相互作用或钙依赖性调节下游通路来介导的。重要的是,概述的Rasal1的分子机制可能有助于正常的神经元发育和突触形成。