, Cells were then stimulated or not with 50 ?m DHPG in Earle's buffer. After a 10-min incubation time in either control or DHPG solution, Dendra2-FMRP-granules were photoconverted through a ×100/ NA 1.4 oil immersion objective for 30 ms using 405-nm laser light (50 mW, 15%). The red photoconverted Dendra2-FMRP was excited using a 561-nm laser light (50 mW, 17%) and two-dimensional-time series (2 Hz) were collected for 10 min. The decrease in red fluorescence from the Dendra2-FMRP photoconverted granules was measured over time using Volocity 6.3 software and data expressed as the percentage of the, the WT or mutated Dendra2-FMRP from were kept in Earle's buffer (25 mM HEPES-Tris, pH 7.4, 140 mM NaCl, 5 mM KCl, 1.8 mM

, A single colony was picked and used to inoculate 25 mL of LB broth supplemented with 50 ?g mL ?1 ampicillin. This was used to inoculate 500 mL of LB and was shaken at 37°C until OD 600 reached 0.8. Cells were then transferred at 20°C and protein synthesis induced by addition of 1 mM IPTG, GST-and His-FMRP production and purification. GST-or His-FMRP (1-160) proteins were produced in E. coli BL21(DE3) cells, p.5

, Cells were disrupted by incubation with 1% lysozyme (Sigma, France) for 30 min at 4°C followed by another 30 min in the presence of 0.1% deoxycholic acid, 10 mM MgCl 2 and 200 ng ?L ?1 DNase. Lysates were then clarified by centrifugation at 10,000×g for 15 min. GST-or His-tagged proteins were purified using either glutathione gel (GE Healthcare) for GST-and GST-FMRP or Nickel resin (Qiagen) for His-fusion proteins. Proteins were then concentrated on Amicon 3-kDa cutoff filters (Millipore) by centrifugation and resuspended in PBS. Concentrations of purified proteins were determined using the BCA protein assay, ETOH, 0.5% NP40, 0.5 M urea) supplemented with 1% protease inhibitor cocktail

, 5 mM MgCl 2 , 0.5% NP40, 0.5 mM DTT, 1% protease inhibitor cocktail) to allow for GST-FMRP/His-FMRP dimerization. Then, 50 ?L of glutathione beads (GE Healthcare) were added to the dimerization mix and incubated at 4°C for 2 h. After five washes in dimerization buffer at 4°C, immobilized GST-FMRP (1-160)-His-FMRP (1-160) dimers were processed for in vitro sumoylation assays, GST-FMRP/His-FMRP dimerization. GST-(control) or GST-FMRP (1-160) fusion proteins (1 ?g) were incubated with an excess of 2 ?g His-FMRP (1-160) for 2 h at 4°C in dimerization buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, vol.2

, After centrifugation for 5 min at 3000×g at 4°C, the supernatant containing the released His-FMRP (1-160) and the pellet containing the remaining immobilized GST-FMRP/His-FMRP dimers were denatured at 95°C for 10 min in, 5× Laemmli buffer containing 7.5% ?-mercaptoethanol and analyzed by immunoblotting with FMRP #2F5-1 antibodies

, Patch clamp experiments were carried out at RT (22-25°C) on mixed cultured cortical/hippocampal neurons obtained from FMRP ?/y mice (four different cultures). FMRP ?/y neurons (18 DIV) were transduced for 24-26 h with attenuated Sindbis virus to express GFP-FMRP WT or the nonsumoylatable GFP-FMRP-K88,130,614R. Patch pipettes displayed a resistance of 4-7 M? and filled with a solution containing

. Cscl, MgCl 2 , 1 Na-GTP, 5 EGTA, and 10 HEPES (pH adjusted to 7.2 with CsOH). The extracellular bathing solution contained, 145 NaCl, 5 KCl

, All data are expressed as mean ± s.e.m. Unpaired t test (Fig. 3i) or non-parametric Mann-Whitney test (Figs. 4c and 5d, f, g) were used to compare medians of two data sets. For spine morphogenesis experiments, values represent means ± s.e.m. Statistical significance for multiple comparison data sets was computed using a one-way analysis of variance with a Bonferroni post-test (Figs. 2b-d, 5e, 7b-d, and Supplementary Fig. 2b-d). Normality for all groups was verified using the Shapiro-Wilk test. According to the Levene variance test, variances were homogenous for the percentage of immature and mature spines (Figs. 2b-d, 7b-d, and Supplementary Fig. 2b-d). For FLIM, data distributions were represented as box and whiskers plots displaying upper and lower quartiles, and maximum and minimum values in addition to median, vol.10

, All relevant data are available from the corresponding author upon reasonable request

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R. Médicale, ) foundations for financial support. We also thank the French Government for the "Investments for the Future, GIS IBiSA (AO 2014) and Région PACA for the Microscopy and Imaging Côte d'Azur (MICA) platform funding