F-Actin-Dependent Regulation of NESH Dynamics in Rat Hippocampal Neurons

Synaptic plasticity is an important feature of neurons essential for learning and memory. Postsynaptic organization and composition are dynamically remodeled in response to diverse synaptic inputs during synaptic plasticity. During this process, the dynamics and localization of postsynaptic proteins are also precisely regulated. NESH/Abi-3 is a member of the Abl interactor (Abi) protein family. Overexpression of NESH is associated with reduced cell motility and tumor metastasis. Strong evidence of a close relationship between NESH and the actin cytoskeleton has been documented. Although earlier studies have shown that NESH is prominently expressed in the brain, its function and characteristics are yet to be established. Data from the present investigation suggest that synaptic localization of NESH in hippocampal neurons is regulated in an F-actin-dependent manner. The dynamic fraction of NESH in the dendritic spine was analyzed using FRAP (fluorescence recovery after photobleaching). Interestingly, F-actin stabilization and disruption significantly affected the mobile fraction of NESH, possibly through altered interactions of NESH with the F-actin. In addition, NESH was synaptically targeted from the dendritic shaft to spine after induction of chemical LTP (long-term potentiation) and the translocation was dependent on F-actin. Our data collectively support the significance of the F-actin cytoskeleton in synaptic targeting of NESH as well as its dynamics

P130Cas Attenuates Epidermal Growth Factor (EGF) Receptor Internalization by Modulating EGF-Triggered Dynamin Phosphorylation

BACKGROUND: Endocytosis controls localization-specific signal transduction via epidermal growth factor receptor (EGFR), as well as downregulation of that receptor. Extracellular matrix (ECM)-integrin coupling induces formation of macromolecular complexes that include EGFR, integrin, Src kinase and p130Cas, resulting in EGFR activation. In addition, cell adhesion to ECM increases EGFR localization at the cell surface and reduces EGFR internalization. The molecular mechanisms involved are not yet well understood. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the molecular mechanism by which p130Cas affects the endocytic regulation of EGFR. Biochemical quantification revealed that cell adhesion to fibronectin (FN) increases total EGFR levels and its phosphorylation, and that p130Cas is required for this process. Measurements of Texas Red-labeled EGF uptake and cell surface EGFR revealed that p130Cas overexpression reduces EGF-induced EGFR internalization, while p130Cas depletion enhances it. In addition, both FN-mediated cell adhesion and p130Cas overexpression reduce EGF-stimulated dynamin phosphorylation, which is necessary for EGF-induced EGFR internalization. Coimmunoprecipitation and GST pull-down assays confirmed the interaction between p130Cas and dynamin. Moreover, a SH3-domain-deleted form of p130Cas, which shows diminished binding to dynamin, inhibits dynamin phosphorylation and EGF uptake less effectively than wild-type p130Cas. CONCLUSIONS/SIGNIFICANCE: Our results show that p130Cas plays an inhibitory role in EGFR internalization via its interaction with dynamin. Given that the EGFR internalization process determines signaling density and specificity in the EGFR pathway, these findings suggest that the interaction between p130Cas and dynamin may regulate EGFR trafficking and signaling in the same manner as other endocytic regulatory proteins related to EGFR endocytosis

NESH Regulates Dendritic Spine Morphology and Synapse Formation

Background: Dendritic spines are small membranous protrusions on the neuronal dendrites that receive synaptic input from axon terminals. Despite their importance for integrating the enormous information flow in the brain, the molecular mechanisms regulating spine morphogenesis are not well understood. NESH/Abi-3 is a member of the Abl interactor (Abi) protein family, and its overexpression is known to reduce cell motility and tumor metastasis. NESH is prominently expressed in the brain, but its function there remains unknown. Methodology/Principal Findings: NESH was strongly expressed in the hippocampus and moderately expressed in the cerebral cortex, cerebellum and striatum, where it co-localized with the postsynaptic proteins PSD95, SPIN90 and F-actin in dendritic spines. Overexpression of NESH reduced numbers of mushroom-type spines and synapse density but increased thin, filopodia-like spines and had no effect on spine density. siRNA knockdown of NESH also reduced mushroom spine numbers and inhibited synapse formation but it increased spine density. The N-terminal region of NESH co-sedimented with filamentous actin (F-actin), which is an essential component of dendritic spines, suggesting this interaction is important for the maturation of dendritic spines. Conclusions/Significance: NESH is a novel F-actin binding protein that likely plays important roles in the regulation o

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 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

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

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

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

SPIN90 phosphorylation modulates spine structure and synaptic function.

The correct rearrangement of postsynaptic components in dendritic spines is important for driving changes of spine structure and synaptic function. SPIN90 plays an essential role in many cellular processes including actin polymerization, endocytosis, growth cone formation and dendritic spine morphogenesis. Here, we demonstrate that SPIN90, which is a binding partner of PSD95 and Shank in spines, is targeted to synapses and leads to enhanced synaptic activity in neurons. We show, using in vitro and in vivo kinase assays, that SPIN90 is tyrosine phosphorylated by Src kinase. SPIN90 that was tyrosine-phosphorylated by Src was targeted to dendritic spines in cultured hippocampal neurons. Moreover, a SPIN90 phospho-deficient mutant was unable to accumulate at dendritic spines whereas SPIN90 WT and a phospho-mimicking mutant were localized at spines and bound PSD95 and Shank with increased efficiency. Consistent with these findings, hippocampal neurons that overexpressed SPIN90 WT or a phospho-mimicking mutant had enlarged spine heads, leading to enhanced postsynaptic function in terms of both amplitude and frequency. Together, our findings show that SPIN90 modulates synaptic activity in neurons as a result of its phosphorylation