目的 探讨Spastin蛋白磷酸化修饰对海马神经元突起生长和分支形成的作用和机制。 方法 用Co-IP及免疫荧光共定位的方法，分析Spastin与微丝（F-actin）是否存在相互作用及二者的结合是否受磷酸化修饰的调控；将Spastin及其磷酸化突变体转染至培养的COS1细胞和大鼠海马神经元，用免疫荧光检测F-actin和突起生长情况。 结果 Co-IP的结果显示Spastin蛋白与微丝存在相互作用，Spastin蛋白S210位点去磷酸化后二者结合增强，磷酸化修饰后二者的结合减弱，与Spastin野生型组差异有统计学意义（P<0.05）；免疫荧光结果显示过表达Spastin促进微丝聚合，S210位点去磷酸化进一步促进微丝聚合，S210位点磷酸化则抑制微丝的聚合作用，与Spastin野生型组差异有统计学意义（P<0.05）。对神经元突起分析的结果显示，过表达Spastin促进神经元突起生长和分支形成，S210位点去磷酸化后此功能进一步增强，S210位点磷酸化则抑制突起生长和分支形成，与Spastin野生型组差异有统计学意义（P<0.05）。 结论 Spastin蛋白S210位点磷酸化修饰通过抑制Spastin与F-actin结合重塑微丝骨架调控神经元突起生长和分支形成。
Abstract
Objective To investigate the role and mechanism of Spastin phosphorylation in the neurites growth and branch formation of hippocampal neurons. Methods Co-IP and immunofluorescence co-localization were used to analyze the interaction between Spastin and F-actin and whether their binding was regulated by phosphorylation modification. Spastin and its phosphorylation mutants were transfected into cultured COS1 cells and rat hippocampal neurons, and immunofluorescence was used to visualize F-actin and neurites. Results Co-IP results showed that Spastin interacted with F-actin, and the binding of Spastin to F-actin was enhanced by dephosphorylation at S210 site of Spastin and weakened by phosphorylation modification, which was statistically different from that of the Spastin wild-type group (P<0.05). Immunofluorescence showed that over-expression of Spastin promoted microfilament polymerization, dephosphorylation of S210 further promoted microfilament polymerization, and phosphorylation of S210 inhibited microfilament polymerization, which was statistically different from that of the wild-type group of Spastin (P<0.05). Analysis of neurites outgrowth showed that over-expression of Spastin enhanced elongation and branching of neurites, which was further enhanced by dephosphorylation of S210, and inhibited by phosphorylation of S210, which was statistically different from that of the wild-type group of Spastin (P<0.05). Conclusions Phosphorylation modification of Spastin at S210 site remodels the microfilament cytoskeleton by inhibiting the binding of Spastin to F-actin, thereby regulating neurite outgrowth and branch formation.
关键词
Spastin； /
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磷酸化； /
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微丝； /
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突起； /
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神经元
Key words
Spastin; /
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Phosphorylation; /
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Microfilament; /
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Neurites; /
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Neuron
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基金
暨南大学医学联合基金项目(YXJC2022003); 广东省自然科学基金面上项目(2021A1515011134)
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