Spy1对CRMP1的磷酸化调节在Sema3A诱导的生长锥塌陷过程中的功能研究

发布时间：2018-04-21 09:52

  本文选题：Spy1 + CRMP1　； 参考：《南通大学》2014年硕士论文

【摘要】：目的验证Spy1与CRMP1的相互作用,分析Spy1对于CRMP1的磷酸化水平调节在Sema3A诱导的生长锥塌陷过程中的影响,探讨两者相互作用与神经元再生之间的关系。方法1.运用免疫共沉淀验证Spy1和CRMP1(collapsin response mediator proteins)之间的相互作用关系;构建Spy1和CRMP1截短突变质粒,转染至工具细胞中,检测其相互作用结构域;同时运用免疫荧光双标检测Spy1和CRMP1在神经元中的共定位情况;免疫共沉淀检测CRMP1与actin的关系;Spy1/CRMP1对于细胞骨架动态性影响。2.Western blot检测Spy1及CRMP1在Sem3A诱导的生长锥塌陷中的表达变化;构建CRMP1点突变质粒,转染原代DRG神经元,分析在Sema3A诱导的生长锥塌陷过程中对于神经元生长锥形态的影响。结果1.在PC12未分化细胞中Spy1和CRMP1存在相互作用;免疫荧光显示Spy1和CRMP1在未分化的PC12中都存在于胞浆;NGF(100ng/ml)刺激PC12未分化细胞分化和原代培养DRG神经元过程中都观察到Spy1和CRMP1在突起末端和边缘的聚集。2.HEK293T工具细胞中发现Spy1域与CRMP1的530~572结构域结合;Spy1可有效的增强CDK5的激酶活性,从而介导对于CRMP1的磷酸化调节作用。3.发现CRMP1与actin存在相互作用;Spy1对CRMP1的磷酸化的增强效应会干扰CRMP1与actin的相互作用,从而影响细胞骨架的动态性,并介导Sema3A的生长锥塌陷效应;体外培养的DRG神经元用Sema3A刺激后发现明显的生长锥塌陷现象;体外siSpy1病毒感染原代DRG神经元并进行Sema3A刺激,发现生长锥的塌陷受到明显的抑制;而siSpy1与CRMP1的拟磷酸化点突变质粒CRMP1T509D或CRMP1S522D共同感染并在Sema3A刺激下,发现生长锥的塌陷现象得到部分回复。结论1.Spy1与CRMP1存在相互作用,Spy1可通过增强CDK5的激酶活性从而增强对CRMP1的磷酸化调节。2.CRMP1与actin存在相互作用;而CRMP1的高磷酸化水平会干扰CRMP1与actin的相互作用,从而影响细胞骨架的动态性,介导Sema3A诱导的生长锥塌陷效应。3.CRMP1的磷酸化点T509和S522介导了Sema3A诱导的生长锥塌陷效应。
[Abstract]:Aim to verify the interaction between Spy1 and CRMP1, to analyze the effect of Spy1 on the phosphorylation of CRMP1 in the process of Sema3A induced growth cone collapse, and to explore the relationship between the interaction and neuronal regeneration. Method 1. The interaction relationship between Spy1 and CRMP1(collapsin response mediator proteins was verified by immunoprecipitation, and the truncated mutant plasmids of Spy1 and CRMP1 were constructed and transfected into tool cells to detect their interaction domains. At the same time, the co-localization of Spy1 and CRMP1 in neurons was detected by immunofluorescence double labeling, and the relationship between CRMP1 and actin was detected by immunoprecipitation. The effect of Spy1 / CRMP1 on cytoskeleton dynamics was detected. 2. Western blot was used to detect the expression of Spy1 and CRMP1 in Sem3A induced growth cone collapse. CRMP1 point mutation plasmid was constructed and transfected into primary DRG neurons. The effect of Sema3A induced growth cone collapse on the morphology of neuron growth cone was analyzed. Result 1. There was interaction between Spy1 and CRMP1 in PC12 undifferentiated cells. Immunofluorescence showed that both Spy1 and CRMP1 were present in undifferentiated PC12 (100 ng / ml) stimulated PC12 undifferentiated cell differentiation and primary culture of DRG neurons. Both Spy1 and CRMP1 were observed in the aggregation of end and edge of protuberance. 2. HEK293T tool cells. The binding of Spy1 domain and CRMP1 530t572 domain to Spy1 can effectively enhance the activity of CDK5 kinase. Thus mediates the phosphorylation regulation of CRMP1. It was found that there was interaction between CRMP1 and actin. The enhancement effect of Spy1 on the phosphorylation of CRMP1 would interfere with the interaction between CRMP1 and actin, thus affecting the dynamics of cytoskeleton and mediating the collapse of Sema3A growth cone. DRG neurons cultured in vitro showed obvious collapse of growth cone after stimulation with Sema3A, and siSpy1 virus infected primary DRG neurons and stimulated with Sema3A in vitro, and found that the collapse of growth cone was obviously inhibited. However, siSpy1 and CRMP1 were co-infected with CRMP1T509D or CRMP1S522D, and under the stimulation of Sema3A, the collapse of growth cone was partially recovered. Conclusion there is interaction between 1.Spy1 and CRMP1. Spy1 can enhance the phosphorylation regulation of CRMP1 by enhancing the activity of CDK5 kinase. 2. CRMP1 interacts with actin, while the high phosphorylation level of CRMP1 interferes with the interaction between CRMP1 and actin. Therefore, the cytoskeleton dynamics was affected, and the growth cone collapse induced by Sema3A was mediated. 3. The phosphorylation point T509 and S522 of CRMP1 mediated the collapse of growth cone induced by Sema3A.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R745
，

本文编号：1781922