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.
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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