Article Figures & Data
Figures
- Figure 1.
α-Chimerin and NR2A colocalize in hippocampal neurons. A, B, Cultured hippocampal neurons (24 DIV) were cotransfected with α1-chimerin (α1-chim)-CFP fusion and untagged NR2A and stained by immunofluorescence 6 h after transfection for NR2A (green) and CFP (red). The merged image shows colocalization of α1-chimerin and NR2A in dendritic spines. Scale bar, 5 μm. C, Cultured hippocampal neurons were cotransfected with α1-chimerin-CFP fusion and NR2A-YFP and imaged 24 h later. A line profile through a dendritic segment shows that increased α1-chimerin-CFP (red) fluorescence intensity correlates with increased NR2A-YFP (green) fluorescence intensity.
- Figure 2.
Phorbol ester translocates α1-chimerin to the plasma membrane. A, Cultured hippocampal neurons (14 DIV) transfected with α1-chimerin-CFP and imaged before (A1) and after (A2) treatment with PMA (300 nm) for 15 min. B, A time course of a 14 DIV hippocampal neuron transfected with α1-chimerin-CFP showing protein translocation after PMA (300 nm) treatment.
- Figure 3.
α-Chimerin interacts with NR2A. A, Lysates from HEK293T cells transfected with NR2A and α1-chimerin-CFP (α1-chim-CFP) were immunoprecipitated with anti-NR2A antibodies in the presence and absence of PMA (300 nm).α1-Chimerin-CFP and NR2A in the immunoprecipitates and lysates (lys) were detected by immunoblotting. PMA treatment increased the amount of α1-chimerin in the immunoprecipitate. The control lanes show the adequacy of the wash step by omitting the IP antibody. B, Association of α1-chimerin (α-Chim) and NR2A in rat hippocampal neuron synaptosomes. The solubilized crude synaptosomal fraction was immunoprecipitated with an anti-NR2A antibody in the presence and absence of PMA (300 nm). The two α-chimerin splice variants and NR2A in the immunoprecipitate and lysate were detected by immunoblotting. The binding of both α1- and α2-chimerin to NR2A was increased after PMA treatment.
- Figure 4.
Interaction of α1-chimerin mutants with NR2A. A, Structure of α1-chimerin mutants in eCFP-C1 vector. Δ27 and Δ4 mutants are C-terminal truncations of α1-chimerin. R179G renders the Rac1 GAP domain of α1-chimerin GTPase inactive. B, Lysates from HEK293T cells transfected with NR2A, and one of the α1-chimerin mutants (α1-chim-CFP) or eCFP alone was immunoprecipitated using an anti-NR2A antibody in the presence and absence of PMA (300 nm). NR2A and the α1-chimerin mutants were detected in the immunoprecipitate and lysate (lys) by immunoblotting. The α1-chimerin Δ27 C-terminal truncation mutant loses its binding affinity for NR2A. Wt, Wild type.
- Figure 5.
Overexpression of α1-chimerin decreases dendritic spine density. A, Cultured hippocampal neurons (21 DIV) were transfected with α1-chimerin and eYFP-N1 or eYFP-N1 alone and imaged for YFP 3 d later. Neurons overexpressing α1-chimerin had fewer spines (0.85 ± 0.0892 spines per 10 μm) than those expressing YFP alone (4.66 ± 0.28 spines per 10 μm). B, α1-Chimerin (α1-chim) overexpression reduces spine density in previously imaged neurons. Cultured hippocampal neurons were transfected with eYFP-N1 at 21 DIV and imaged, and the x, y coordinates of each neuron were recorded. At 24 DIV, the same dish of neurons was transfected with α1-chimerin-CFP, and previously characterized neurons were inspected for α1-chimerin expression. B1, Dendrite of a neuron that was transfected with eYFP-N1 at 21 DIV before overexpression of α1-chimerin. B2, The same dendrite of the neuron shown in B1, now expressing both YFP and α1-chimerin-CFP. Overexpression of α1-chimerin eliminates preexisting spines. Scale bars, 5 μm.
- Figure 6.
α1-Chimerin antagonizes the effects of Rac1 on spine density. A, Cultured hippocampal neurons (21 DIV) were transfected with eYFP, eYFP plus Rac1, eYFP plus Rac1 plus α1-chimerin, or eYFP plus dominant-negative (DN) Rac1 (N17). Neurons were imaged for YFP 3 d later. Spine density is significantly reduced in both Rac1 plus α1-chimerin and dominant-negative Rac1 (0.79 ± 0.112 spines per 10 μm). Scale bar, 5 μm. B, Quantification of spine density in neurons transfected with the constructs pictured in A. C, Hippocampal neurons (21 DIV) cotransfected with eYFP and constitutively active Rac1 (I115) developed large dendritic lamellipodia. Spines can no longer be reliably identified in these neurons. Similar lamellipodia were present when eYFP, active Rac1 (I115), and α1-chimerin were cotransfected into 21 DIV hippocampal neurons.
- Figure 7.
The effects of α1-chimerin on spines depend on its Rac1 GAP activity and its ability to bind NR2A. A, Neurons were transfected with eYFP alone or with both eYFP and wild-type α1-chimerin (WT), α1-chimerin R179G GTPase activation mutant (R179G), α1-chimerin Δ4 mutant, or α1-chimerin Δ27 mutant. YFP images of representative dendrites are shown for each condition. Overexpression of the GTPase activation mutant no longer affects spine density. The Δ27 mutant, which has much lower affinity for NR2A than wild-type α1-chimerin (Fig. 4 B) also has lost its ability to reduce spine density. Scale bar, 5 μm. B, Quantification of spine density in neurons transfected with the constructs pictured to the left. Spine density is significantly reduced only for wild-type α1-chimerin and its Δ4 mutant, whereas the GAP-inactive mutation and the Δ27 deletion mutant have no effect on spine density. α1-chim-CFP, α1-Chimerin-CFP.
- Figure 8.
Downregulation of α1-chimerin increases spine density. A, Cotransfection of hippocampal neurons (10 DIV) with eCFP and α1-chimerin siRNA expression plasmid. Cells were immunostained at 14 DIV against α-chimerin. Note the decreased α-chimerin signal in the transfected neuron (arrow). B, Hippocampal neurons cotransfected with eYFP and random siRNA (left) or eYFP and α1-chimerin siRNA (right). Neurons expressing the α-chimerin-specific siRNA show increased spine density (8.31 ± 1.23 spines per 10 μm; p < 0.01) when compared with those expressing a random siRNA construct (5.56 ± 0.54 spines per 10 μm). Scale bar, 5 μm.
Additional Files
Supplemental data
Files in this Data Supplement:
- supplemental material - Supplemental Figure 1. ?-chimaerin and NR2A interact in a yeast-two hybrid assay. A. Domain structure of ?-chimaerin and the NMDA subunit C-terminal domains used to confirm the yeast-two-hybrid result. B. Yeast strain AH109 co-transformed with ?-chimaerin and either one of the three NR2A -terminal domains, the NR1 C-terminal domain, or empty bait vector (pGBKT7) as specified in the diagram on the right. Growth occurs only when ?-chimaerin is co-transformed with the second NR2A domain or the full-length NR2A C-terminal domain.
- supplemental material - Supplemental Figure 2. Model of ? �chimaerin�s proposed role in spine remodeling. Spine shape and density is thought to be responsive to synaptic signaling including NMDA receptor dependent LTP and LTD and mGluR1 dependent LTD. Spines on neurons undergoing LTP tend to increase in size and number while those undergoing LTD become smaller. Rho GTPase regulators like Tiam1 may localize to developing synapses by binding to the NMDA receptor subunit NR2B, the predominant subunit present at developing synapses. Once bound, they may activate Rac1 and promote the formation of new spines. a-chimaerin, on the other hand, may be activated by mGluRs at synapses undergoing LTD. Upon glutamate binding to mGluR1, Gaq is released and activates PLC which leads to the production of diacylglycerol (DAG). DAG recruits a-chimaerin to the neuronal membrane where it binds NR2A, the predominant subunit at mature excitatory synapses. Active a-chimaerin, bound to synaptic NMDA receptors, locally inactivates Rac1 and promotes spine contraction.
