The C-terminal region of FMRP contains evolutionary conserved nucleolar localization signals.

<p>(A) Domain organization and motifs of FMRP. Schematic diagram of FMRP architectures highlights major domains and motifs. FCT, FMRP C-terminus; KH1 and KH2, tandem K (described first in the hnRNP K protein) homology domain; NES, nuclear export signal; NLS, nuclear localization signal; NoLS, nucleolar localization signal; PPID, protein-protein interaction domain; RGG, arginine-glycine-glycine region; P, phosphorylation sites; Tud1 and Tud2, tandem Tudor (also called Agenet) domains. The C-terminal region (Cterm; aa 444–632) of FMRP contains two NoLSs, identified in this study. Two further FRMP fragment used were Nterm (1–218) and a Central region (212–425). (B) Overexpression of the Cterm wild-type (wt) and its variants on HeLa cells. Cterm 1: QKKEK changed to EEEeE; Cterm 2: RRGDGRRR changed to EEgdgEEE; Cterm 3: RR changed to EE; Cterm 1+2: a combination of Cterm 1 and 2 mutations; Cterm 1+3: a combination of Cterm 1 and 3 mutations. Cterm 2 and Cterm 1+2 revealed a change in protein mobility (**) as compared to the wild-type and the other variants (*). (C) NoLS prediction of FMRP Cterm using the NoLS predictor program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091465#pone.0091465-Scott1" target="_blank">[58]</a>. Graph shows the probability of NoLS distribution (represented by score) plotted against the amino acid sequence of FMRP Cterm (444–632). Three motifs and critical positively charged residues are marked blue. (D) Multiple sequence alignment of the three predicted NoLS motifs 1, 2, and 3 of FMRP Cterm from different species (upper panel) as well as FMRP transcripts and homologous proteins (lower panel). Basic residues (blue), which are changed to glutamic acids (red) are highlighted. Upper panel: FMRP sequences from different species are human (accession number 544328), orangutan (197102198), rat (30794228), frog (53749722) and zebrafish (23308667). Lower panel: FMRP transcripts and homologous proteins are transcript 6 (297374777), 7 (297374779), 9 (297374791) and 12 (297374789) as well as FXR1P (61835148) and FXR2P (259013556). (E) Nucleolar localization of FMRP. cLSM images of HeLa cells transfected with FMRP fl, Nterm, Central, Cterm (wt) and Cterm variants (anti-flag; green channel) costained with endogenous nucleolin (anti-nucleolin; red channel) and DNA (DAPI; blue channel) revealed that Cterm (wt), (1), (2), (3) and (1+3) colocalize with nucleolin in the nucleolus. In contrast this colocalization was absent in the case of Cterm (1+2). Cytoplasmic distribution of FMRP Nterm and the subnuclear distribution of endogenous nucleolin are highlighted by arrows. Scale bar: 10 μm.</p

FMRP shows a diverse subcellular distribution pattern in HeLa cells as revealed by subcellular fractionation analysis.

<p>(A) Experimental cell fractionation procedure employing several differential centrifugation steps. Cells were fractionated into six distinct fractions, including heavy membrane (plasma membrane and rough endoplasmic reticulum), light membrane (polysomes, golgi apparatus, smooth endoplasmic reticulum), cytoplasm (cytoplasm and lysosomes), enriched nuclear membrane (containing rough endoplasmic reticulum), nucleoplasm, and nucleoli. S, supernatant; P, pellet. (B) FMRP is largely absent in the cytoplasm and nucleoplasm and predominantly localizes to solid compartments. The protein concentrations were normalized in all fractions with exception of the nucleoplasm due its low protein content as compared to the other fractions. In each lane, 5 μg proteins were loaded except for the nucleoplasm, where one μg was used. In addition to FMRP and its binding partner CYFIP, the fractions were analyzed by using different subcellular marker, including Gα_q/11, Na⁺/K⁺-ATPase and Rac1 (plasma membrane), EEA2 (endosomes), GAPDH (cytoplasm), eIF5 and RPLP0 (ribosomes and rough ER). Nuclear markers included histone H3 and lamin B1. Nucleolin was used as nucleolar marker. (C) Detection of FMRP in mitochondria. The presence of FMRP in isolated mitochondrial fraction was analyzed by SDS-PAGE and immunoblotting, using antibodies against FMRP, two mitochondrial proteins MTCO2 and ACAT1, the cytosolic GAPDH as well as the nuclear proteins lamin B1, histone H3 and nucleolin. Equal protein amounts of the mitochondrial fraction and the total cell lysate were used.</p

FMRP is localized at various intracellular sites in HeLa cells.

<p>Confocal laser scanning microscopy (cLSM) images of HeLa cells depicting endogenous FMRP (green channel) costained with various cytosolic (A) and nuclear (B) markers (red channel), including antibodies against CYFIP2, RPLP0 (ribosomal proteins), nucleolin (nucleolar marker), MTCO2 (mitochondrial protein), NUP62 (nucleoporins), lamin B1 (nuclear intermediate filament proteins), and calreticulin (endoplasmic reticulum marker). Detection of Na⁺/K⁺-ATPase and phalloidin staining were used to detect the cellular membrane and F-actin, respectively. DNA was stained by using DAPI (blue channel). Boxed areas in the merged panels depict enlarged areas of interest. Scale bar: 10 μm.</p

FMRP and nucleolin interact in both cytosolic high molecular weight and nuclear low molecular weight complexes.

<p>(A, B) Native FMRP protein complexes were fractionated by loading the light membrane (A) and nucleolar (B) fractions on a superose 6 size exclusion chromatography column. The absorbance of the column eluent at 280 nm (A₂₈₀) was plotted against the elution volume (ml). Different FMRP binding partners and markers are shown, including histone H3 and GAPDH as negative controls in the endomembrane and nucleolar fractions, respectively. The elution positions of standard proteins employed include thyroglobuline (669 kDa), ferritin (440 kDa), aldolase (158 kDa), ovalbumin (75 kDa), carbonic anhydrase (29 kDa), ribonuclease (13.7 kDa), and aprotinin (6.5 kDa). The peak fractions, as indicated by a solid line, were subjected to SDS-PAGE and immunoblotting using antibodies against FMRP (71 kDa), nucleolin (76 kDa), CYFIP2 (146 kDa), RPLP0 (34 kDa) and eIF5 (58 kDa) LM, light membrane; Nu, nucleoli. The molecular mass of the peak fractions is indicated above the peaks. (C) Interaction of FMRP with CYFIP, nucleolin, eIF5, and RPLP0 as analyzed by co-immunoprecipitation. Endogenous FMRP was immunoprecipitated from HeLa cell lysates using an anti-FMRP antibody before and after RNase treatment. FMRP co-precipitated with nucleolin, RPLP0, eIF5, and CYFIP2. Interaction with the latter two proteins was sensitive to RNase treatment. Proteins were visualized by using antibodies against FMRP, nucleolin, eIF5, CYFIP2 and RPLP0. N-WASP and GAPDH were used as a negative IP controls. IP, immunoprecipitation; TCL, total cell lysate. (D) Direct interaction between FMRP and nucleolin. GST pull-down experiments were conducted by mixing bacterial lysate expressing His-tagged FMRP fl (upper panel) or FMRP Nterm (lower panel) with different GST-fused nucleolin proteins (RRM1&2, aa 284–466; RRM3&4, aa 467–644; RRM3&4-RGG, aa 499–710; RGG, aa 645–710) immobilized on GSH sepharose beads. Proteins retained on the beads were resolved by SDS-PAGE and processed for Western blot using a monoclonal antibody against FMRP. Mixed samples before performing pulldown (PD) analysis were used as input control. (E) Low-affinity interaction between the FMRP Nterm and the nucleolin RGG. Fluorescence polarization assay was used as a tool for monitoring the interaction of the FMRP Nterm (increasing concentrations as indicated) with the IAEDANS-labeled fluorescent RGG (0.5 μM) (open circles). As negative controls, FMRP Nterm was titrated into IAEDANS alone (0.5 μM) (closed circles). The inset depicts the displacement of FMRP Nterm from IAEDANS-labeled fluorescent RGG by increasing concentrations of unlabeled RGG and the synthetic peptide construct 5(KPR)TASP.</p

The PI3K-AKT signaling is altered in Ptpn11^D61Y brains.

<p>(A) Representative Western blots of forebrain homogenate from control and Ptpn11^D61Y mice. The phosphorylation level of AKT at the residues Thr308 and Ser473 are reduced, while the phosphorylation of S6K is increased. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006684#pgen.1006684.s008" target="_blank">S8 Fig</a> for representative images of all proteins analyzed. <b>(B)</b> Quantification of the Western blots exemplified in <b>A</b>. Data is shown as mean ± SEM and normalized to the expression in controls (n = 3 animals per genotype, unpaired t-test; *p<0.05; **, p<0.01).</p

Analysis of DEGs in hippocampi of control and Ptpn11^D61Y mice in the basal state and after the stimulation of neuronal activity.

<p><b>(A)</b> The heat maps show the expression levels of DEG transcripts for each analyzed dataset. Three biological replicates (columns) per condition are shown for all DEGs (rows). The color code in a logarithmic scale is given for the signal intensities of the DEGs and indicates a low (in blue) or high (red) expression. <b>(B)</b> The Venn diagram shows the number of DEGs in each dataset. The intersections indicate the number of genes regulated in two or more datasets. <b>(C)</b> The table shows the number of differentially expressed mRNAs and miRNAs as well as the direction of their regulation in each dataset. <b>(D-F)</b> Plots show the inter-dependency of the expressional regulations of the DEGs that are commonly regulated between the datasets. Each data point represents one DEG; the x- and y-axis indicate the level of regulation as fold change in a logarithmic scale in the dataset. The correlation coefficient (r) and p-value are indicated in the graphs. The black lines show the best fits; the dashed lines indicate the 95% confidence intervals.</p

Activity-induced increase of pERK is abolished in Ptpn11^D61Y neurons and can be restored upon MEK1 inhibition.

<p>Staining <b>(A)</b> and quantification <b>(B)</b> of pERK in nuclei (marked by DAPI) of excitatory neurons revealed elevated basal nuclear pERK levels in Ptpn11^D61Y neurons. The basal levels of nuclear pERK significantly differ between the genotypes (unpaired t-test, ###p≤0.001). The stimulation of neuronal activity induces a rapid increase in the nuclear pERK level (in relation to the basal levels) in control neurons but not in those from Ptpn11^D61Y mice. The inhibition of MEK1 using SL327 for 24 h prior to the stimulation normalized the elevated basal pERK in nuclei of Ptpn11^D61Y neurons and fully restored the activity-induced increase of nuclear pERK. The identical treatment affected neither the basal nuclear pERK levels nor their stimulation-induced increase in control neurons. Data are presented as mean ± SEM; numbers in columns indicate the numbers of analyzed cells. Significance is assessed using unpaired t-test and one-way ANOVA followed by Bonferroni´s multiple comparison test (**p≤0.01, ***p≤0.001).</p

Neuronal activity-induced phosphorylation of ERK is disturbed in Ptpn11^D61Y.

<p><b>(A)</b> Exemplary photograph of an acute slice used for experiments. <b>(B)</b> Western blot of lysates from control and Ptpn11^D61Y acute slices treated with 4AP/Bic (<b>+</b>) or vehicle (<b>-</b>) for 10 minutes and probed with antibodies against pERK, ERK and β-III-tubulin. The latter was used as a loading control. <b>(C-E)</b> Quantification of the Western blot experiment as exemplified in B is shown. The stimulation of control slices led to a significant increase of the pERK level <b>(C, D)</b>. The basal pERK level was increased in Ptpn11^D61Y slices compared to controls. No further increase of pERK immunoreactivity could be detected upon stimulation of neuronal activity <b>(C, D)</b>. Note the increased total expression of ERK in the slices from Ptpn11^D61Y in the basal state and upon stimulation <b>(E)</b>. <b>(F-H)</b> The quantification of ERK phosphorylation in the nuclear fraction prepared from forebrains indicates an increase in the pERK level in the samples from Ptpn11^D61Y animals as compared to controls. Data are shown as mean ± SEM and analyzed using either one-way ANOVA followed by Bonferroni´s multiple comparison test or unpaired t-test (*p≤0.05, ***p≤0.001). The number of replicates from a total of three independent experiments is indicated in the columns of the graph.</p

Synaptic expression and surface trafficking of glutamate receptors is affected in Ptpn11^D61Y.

<p><b>(A)</b> Representative examples of the staining of synaptic surface (left) and total (right) glutamate receptors containing GluA1 or GluN2B subunit in neurons from control and from Ptpn11^D61Y. Excitatory synapses were stained for Homer1 and Bassoon. <b>(B, C)</b> Synaptic IF intensity and density (puncta per 20 μm of proximal dendrite) of surface <b>(B)</b> and total <b>(C)</b> staining for all tested receptor subunits are shown. Values are normalized to the respective value in control, represented by the dashed line in the graphs. Note the reduced surface expression of GluA2 and GluN2B and the decrease of the overall expression of GluA1 in Ptpn11^D61Y neurons as compared to controls. All numerical values and statistics are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006684#pgen.1006684.s009" target="_blank">S1 Table</a>. <b>(D, E)</b> The rate of internalization of the GluA1-containing AMPARs is reduced in Ptpn11^D61Y cells. Scale bars: 5 μm. Data are presented as mean ± SEM and analyzed using unpaired t-test (*p≤0.05, **p≤0.01, ***p≤0.001). Numbers in columns indicate the number of cells analyzed.</p

Activity-induced BDNF expression is affected in Ptpn11^D61Y neurons.

<p><b>(A)</b> Representative images of neurons in low-density cultures infected with the lentivirus containing the BDNFpI+II EGFP reporter in basal conditions and upon treatment with 4AP/Bic for 30 min. Neurons are stained with antibodies against GFP to enhance the intrinsic EGFP signal, MAP2 as a neuronal marker, and DAPI. <b>(B)</b> Schema of the activity reporter, in which the BDNF promoters I and II drive the expression of EGFP. The cAMP response element (CRE) is depicted. <b>(C)</b> Quantification of the GFP IF that was measured in the nuclei of control and Ptpn11^D61Y neurons. The reporter signal increased significantly upon stimulation in control but not in Ptpn11^D61Y neurons. Data are shown as mean ± SEM and analyzed using one-way ANOVA followed by Bonferroni´s multiple comparison test (*p≤0.05) Scale bar: 10 μm.</p