Synaptic translocation of NESH via LTP induction.

<p>The glycine-induced chemical LTP (long-term potentiation) method was used to mimic physiological LTP. (A) Chemical LTP (cLTP) was induced in hippocampal neurons at 16–18 DIV, and the level of phospho-PAK assessed to ascertain cLTP induction. (B) After cLTP induction, the Triton X-100-insoluble fraction was extracted from hippocampal neurons, and the association between NESH and cytoskeleton or synaptic site examined using immunoblot analysis. (C) The band intensities were quantified and normalized by loading control (α-tubulin). (D) Synaptic translocation of NESH was examined during LTP. Hippocampal neurons at 10–12 DIV were transfected with GFP-NESH (or GFP). After cLTP induction at 16–18 DIV, transfected neurons were fixed and stained with Alexa 594-conjugated phalloidin, and NESH localization examined. GFP served as the control. (E) Analysis of the fluorescence intensity ratio in dendritic spine vs. shaft from data obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034514#pone-0034514-g006" target="_blank">Fig. 6D</a> (N = 12–16 neurons for each condition). Data are presented as means ± SEM. *p<0.05, **p<0.01, ***p<0.001.</p

F-actin-dependent synaptic translocation of NESH in hippocampal neurons.

<p>(A) The effect of F-actin stabilization on localization of NESH was investigated. Hippocampal neurons were co-transfected with GFP-NESH (or GFP) and pLifeact-TagRFP at 10–12 DIV. pLifeact-TagRFP was employed to image the F-actin cytoskeleton within cells. Transfected neurons at 16–18 DIV were treated with jasplakinolide (5 µM for 10 min), fixed, and imaged. Colocalization between NESH and F-actin is indicated with white arrows in the merged image. (B) Quantitative analysis of the intensity ratio of spines vs. shafts from data obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034514#pone-0034514-g001" target="_blank">Fig. 1A</a> (N = 16 neurons for each condition). Data are presented as means ± SEM. ***p<0.001.</p

Effect of F-actin stabilization on NESH mobility in dendritic spines.

<p>FRAP analysis was performed to examine the effect of F-actin stabilization on NESH mobility. Hippocampal neurons at 10–12 DIV were transfected with GFP-NESH, GFP, GFP-actin, GFP-PSD95 or GFP-Homer1c, and subjected to FRAP analysis at 16–18 DIV. (A, F) Recovery curves of GFP-NESH in non-treated (control) and jasplakinolide-treated neurons (mobile fractions: 43.9±2.4% at 400 s for control, 13.6±2.8% at 400 s for jasplakinolide). (B, F) Recovery curves of GFP (mobile fractions: 92.7±8.0% at 400 s for control, 83.3±1.3% at 400 s for jasplakinolide). (C, F) Recovery curves of GFP-actin (mobile fractions: 98.6±8.7% at 400 s for control, 0.2±0.1% at 400 s for jasplakinolide). (D–F) Recovery curves and mobile fractions of the scaffolding proteins, PSD95 and Homer1c (control: 16.7±5.2% at 400 s for GFP-PSD95, 14.8±6.6% at 400 s for GFP-Homer1c, jasplakinolide: 12.0±2.7% at 400 s for GFP-PSD95, 7.0±4.2% at 400 s for GFP-Homer1c). (F) Analysis of the mobile/immobile fractions from data obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034514#pone-0034514-g003" target="_blank">Fig. 3A–E</a> (N = 15 for each condition, data from three to five independent experiments). Data are presented as means ± SEM. **p<0.01.</p

FRAP analysis of NESH dynamics in dendritic spines.

<p>(A) To investigate the dynamics of NESH in a single spine, the FRAP (fluorescence recovery after photobleaching) assay was performed. Hippocampal neurons at 10–12 DIV were transfected with GFP-NESH and subjected to FRAP imaging at 16–18 DIV. A single spine of GFP-NESH-transfected neurons was set to ROI (region of interest) and bleached for 7 s with an Ar 488 laser, and recovery of GFP-NESH observed at intervals of 10 s over a time-course of 5 min. (B) Recovery curve of GFP-NESH from data obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034514#pone-0034514-g002" target="_blank">Fig. 2A</a>. NESH fluorescence was slowly recovered (up to ∼40%) for 5 min (N = 15, data from three to five independent experiments). (C) Hippocampal neurons at 10–12 DIV were transfected with GFP, GFP-actin, GFP-PSD95 or GFP-Homer1c, and used for FRAP imaging at 16–18 DIV. NESH mobility was compared with that of other proteins using FRAP (N = 15 for each protein, data from three to five independent experiments). (D) Analysis of mobile/immobile fractions from data obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034514#pone-0034514-g002" target="_blank">Fig. 2B and C</a>. F_end: fluorescence at the end time-point, F_post: fluorescence right after photobleaching, F_pre: fluorescence before photobleaching, M_f: mobile fraction, I_f: immobile fraction. Data are presented as means ± SEM.</p

F-actin-dependent synaptic translocation of NESH after LTP induction.

<p>(A) Hippocampal neurons at 10–12 DIV were transfected with GFP-NESH (or GFP). To determine the importance of the F-actin cytoskeleton in NESH translocation during cLTP, transfected neurons at 16–18 DIV were treated with latrunculin A (5 µM for 10 min), and cLTP was subsequently induced. Following fixing of neurons, NESH localization was examined. (B) Analysis of the intensity ratio (spine vs. shaft) from data obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034514#pone-0034514-g007" target="_blank">Fig. 7A</a> (N = 11–13 neurons for each condition). Data are presented as means ± SEM. **p<0.01.</p

Effect of F-actin disruption on mobility of NESH.

<p>To examine the effect of F-actin disruption on NESH mobility in the dendritic spine, FRAP analyses were performed using hippocampal neurons at 16–18 DIV that were transfected with GFP-NESH, GFP, GFP-actin, GFP-PSD95 and GFP-Homer1c at 10–12 DIV. F-actin disruption was induced by treating with latrunculin A (5 µM for 10 min). (A, F) Recovery curves of GFP-NESH in non-treated (control) and latrunculin A-treated neurons (mobile fractions: 45.6±1.9% at 400 s for control, 24.9±3.6% at 400 s for latrunculin A), suggesting that dynamic actin remodeling is crucial for the mobility and dynamics of NESH. (B, F) Recovery curves of GFP (mobile fractions: 98.2±2.2% at 400 s for control, 95.4±10.3% at 400 s for latrunculin A) (C, F) Recovery curves of GFP-actin (mobile fractions: 98.1±2.6% at 400 s for control, 54.2±11.4% at 400 s for latrunculin A) (D–F) Recovery curves and mobile fractions of scaffold proteins, GFP-PSD95 and GFP-Homer1c, showed no significant differences. (F) Analysis of mobile/immobile fractions from data obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034514#pone-0034514-g004" target="_blank">Fig. 4A–E</a> (N = 15 for each condition, data from three to five independent experiments). Data are presented as means ± SEM. *p<0.05.</p

NESH knockdown causes abnormal morphological changes in dendritic spines.

<p>(A) HEK 293T cells were co-transfected with GFP-NESH and siRNAs and then immunoblotted with anti-GFP antibody after incubation for 48–72 h. (B) Cultured hippocampal neurons were transfected with NESH siRNAs at 10–12 DIV, and NESH knockdown was evaluated by immunoblotting at 16–18 DIV. (C) Knockdown of NESH by si591 was confirmed with immunofluorescence assay in hippocampal neurons. GFP was co-transfected with siRNAs to visualize transfected neurons. White arrows indicate untransfected neurons and arrow heads indicate transfected neurons. (D–H) Morphometric analyses were performed to examine the effects of NESH knockdown in hippocampal neurons (n = 18 neurons for control; n = 19 neurons for NESH siRNA). Hippocampal neurons were transfected with control (scrambled siRNA) or NESH siRNA (si591) at 10–12 DIV and fixed at 16–18 DIV. GFP was co-transfected to visualize dendritic spines. The images were acquired using an Olympus IX81 fluorescence microscope. (D) Fluorescence images of neurons transfected with NESH siRNA or scrambled siRNA (control). (E) Spine density in NESH knockdown and control neurons. (F) Densities of the four types of dendritic spines (mushroom, thin, stubby or branched). (G) Spine head width. (H) Spine length. Data are presented as means ± SEM. *p<0.05, **p<0.01, ***p<0.001.</p

NESH knockdown reduces synapse formation and affects the postsynaptic apparatus.

<p>(A) Hippocampal neurons were transfected with the control (scrambled siRNA) or NESH siRNA at 10–12 DIV and stained with anti-VAMP2 antibody, anti-GluR1 antibody or Alexa Fluor 594-conjugated phalloidin at 16–18 DIV. GFP was co-transfected with siRNAs to visualize dendritic spines. (B) Synapse formation per µm was analyzed in NESH knockdown neurons and compared with control (n = 15 for control; n = 19 for NESH siRNA). (C) Numbers of GluR1 cluster per µm on spines (n = 17 for control; n = 15 for NESH siRNA). (D) F-actin fluorescence intensity ratios (spine vs. shaft; n = 15 for control and NESH siRNA). Data are presented as means ± SEM. *p<0.05, **p<0.01.</p

Overexpression of NESH prevents synapse formation in hippocampal neurons.

<p>(A) Cultured hippocampal neurons were co-transfected with GFP and myc-NESH at 10–12 DIV and fixed at 16–18 DIV. Empty vector was used as a control, and GFP was used to visualize dendritic spines. The fixed neurons were stained with anti-VAMP2 (presynaptic marker) antibody, anti-GluR1 (subunit of AMPA receptor) antibody or Alexa Fluor 594-conjugated phalloidin. (B) Synaptic densities were analyzed by counting the dendritic spines contacting presynapses marked by VAMP2 staining. (C) GluR1 clusters on dendritic spines were measured in neurons overexpressing NESH and compared with control. (D) F-actin fluorescence intensity ratios (spine vs. shaft). Data were obtained from three independent experiments; n = 20 each for control and NESH-overexpressing neurons. Data are presented as means ± SEM. *p<0.05, **p<0.01.</p

NESH interacts directly with filamentous actin via its N-terminal region, but not with monomeric actin.

<p>(A) Schematic diagram showing representations of full-length NESH (amino acids 1–367), N-term (N-terminal half, amino acids 1–229) and C-term (C-terminal half, amino acid 221–367). (B) GST pull-down assays were performed to verify the interaction between NESH and monomer G-actin. GST-fused NESH proteins were incubated with purified monomeric G-actin and then pulled down with glutathione Sepharose beads, after which the bound proteins were detected with anti-actin antibody. GST-SPIN90-C-term served as a positive control. (C) F-actin co-sedimentation assays. Purified NESH proteins were incubated with polymerized F-actin. After separating the supernatant (S) and pellet (P) by ultracentrifugation, co-sedimented proteins were detected by Coomassie Brilliant Blue staining. (D) NESH N-term and C-term in F-actin co-sedimentation assays. Note that NESH N-term only interacts with F-actin.</p