Activity regulates eEF2K-dependent spine morphogenesis.

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Activity regulates eEF2K-dependent spine morphogenesis. Activity regulates eEF2K-dependent spine morphogenesis. A, Neurons were transfected or not with eEF2K together with GFP on DIV10, treated with TTX or bicuculline on DIV16, fixed on DIV18 and immunostained to reveal eEF2K. Overexpression of eEF2K (lower panels in both treatments) modified spine morphology only in bicuculline-treated neurons. B, COS-7 cells were transfected or not with the indicated amounts of eEF2K or eEF2KSer366Ala (eEF2Kca), and solubilized in Laemmli buffer after 48 h. Levels of eEF2K, P-eEF2 and total eEF2 were analyzed by Western blot. C, Mean (error bars SEM) P-eEF2 levels (normalized against total eEF2 signal) in cells transfected with the indicated amounts of each construct (data collected from four independent experiments). *p < 0.05 (Student's t test). D, Neurons were transfected with an empty vector or eEF2Kca together with GFP on DIV10, fixed on DIV18 and immunostained to reveal eEF2K. eEF2Kca overexpression changed spine morphology even in the absence of stimulation. E–G, Mean length, width and number of dendritic spines (error bars SEM) in neurons transfected with indicated constructs at reported treatments. Over 10 neurons from three independent experiments were measured at each condition; *p < 0.05 versus Bic+eEF2K; #p < 0.05 versus Bic; §p < 0.05 versus vector transfected neurons (ANOVA, Tukey test). Scale bar, 10 μm. Chiara Verpelli et al. J. Neurosci. 2010;30:5830-5842 ©2010 by Society for Neuroscience