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First published online 14 November 2006
doi: 10.1242/jcs.03273

Journal of Cell Science 119, 4986-4993 (2006)
Published by The Company of Biologists 2006
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Sequential implication of the mental retardation proteins ARHGEF6 and PAK3 in spine morphogenesis

Roxanne Nodé-Langlois, Dominique Muller and Bernadett Boda*

Department of Basic Neuroscience, University of Geneva, School of Medicine, 1211 Geneva 4, Switzerland


Figure 1
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  		Fig. 1. Hippocampal expression of ARHGEF6 and localization in postsynaptic densities. (A) Level of expression of ARHGEF6 (filled circles), PAK3 (open circles) and CaMKII (open squares) in the CA1-3 region of the hippocampus, assessed using real-time fluorescence RT-PCR. Reverse transcription was performed with 1 µg of total RNA and a dilution corresponding to 16 ng of total RNA was amplified in a SYBRGreen PCR reaction. Data are mean of triplicate samples. (B) A pyramidal neuron co-transfected with EGFP (green, left, GFP) and HA-tagged ARHGEF6 (red, right, HA-GEF) showing a diffuse cytoplasmic and a punctate dendritic distribution of HA-ARHGEF6. (C) Magnified view of a dendritic branch obtained from a neuron co-transfected with EGFP (green, left, GFP) and HA-ARHGEF6 (red, right, HA-GEF), showing the punctate distribution at the level of spines. (D) A neuron co-transfected with PSD-95-EGFP and HA-ARHGEF6 showing the colocalization of PSD-95-EGFP puncta (PSD-95, green, left) with HA-ARHGEF6 (HAGEF, red, right). (E) Colocalization of synaptophysin immunostaining (green) with HAARHGEF6 puncta (red). Bars, (B) 20 µm; (C-E) 2 µm.

 

Figure 2
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  		Fig. 2. Knockdown of ARHGEF6 using a siRNA transfection approach. (A) Western blot illustrating the level of expression of HA-ARHGEF6 in non-transfected fibroblasts, in fibroblasts transfected with HA-ARHGEF6, and fibroblasts co-transfected with HA-ARHGEF6 and either a non-silencing siRNA or the silencing siR1 and siR2 constructs. (B) Western blot illustrating the absence of interference of the siR1 and siR2 silencing constructs with the closely related ßPix protein (weak band corresponds to a less expressed ßPix isoform). (C) Punctate HA staining (red, left, HA-GEF) of a dendritic segment of a cell co-transfected with EGFP and HAARHGEF6, which contrasts with the HA staining (red, middle, HA-GEF + siR1) obtained from a cell co-transfected with HAARHGEF6, siR1 oligos and EGFP (green, right, GFP). Bar, 2 µm. (D) Quantitative analysis of HA-ARHGEF6 staining showed 89±5% reduction in siR1 co-transfected pyramidal neurons as compared to control cells. Data are mean ± s.e.m., n=4-7; P<0.05.

 

Figure 3
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  		Fig. 3. Absence of spine abnormalities induced by overexpression of ARHGEF6, wild-type PAK3 or constitutively active PAK3. (A) Dendritic segments in cells transfected with EGFP (Ctrl) or cotransfected with EGFP and either wild-type ARHGEF6, wild-type PAK3 (wtPAK3) or constitutively active PAK3 (caPAK3). Bar, 2 µm. (B) Blind quantitative analysis of large mushroom-type spines (spine head diameter >0.7 µm), filopodia or elongated spines (>=1.8 µm in length) show no detectable differences between control, EGFP-transfected cells (open bars) and ARHGEF6 (black bars), wild-type PAK3 (grey bars) or constitutively active PAK3 (dashed bars) cotransfected neurons. Data are mean ± s.e.m., n=5-12.

 

Figure 4
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  		Fig. 4. Spine abnormalities induced by ARHGEF6 knockdown in hippocampal organotypic slice cultures. (A) Dendritic segments in cells transfected with EGFP (Ctrl) or co-transfected with EGFP and either a non-silencing siRNA or silencing siR1 and siR2 constructs. Note the increase in elongated spines and filopodia. Bar, 3 µm. (B) Blind quantitative analysis of large mushroom-type spines (spine head diameter >0.7 µm) shows a significant reduction in siR1 (dark grey bar) and siR2 (grey bar) transfected cells versus EGFP-transfected control neurons (white bar) or non-silencing siRNA transfected cells. (C) Increase in filopodia observed under the same experimental conditions. (D) Increase in elongated spines (>=1.8 µm). Data are mean ± s.e.m., n=6-20; *P<0.05.

 

Figure 5
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  		Fig. 5. Co-transfection with constitutively active PAK3, but not wild-type PAK3 rescues the phenotype induced by ARHGEF6 knockdown. (A) Illustration of dendritic segments obtained from pyramidal neurons transfected with EGFP (Ctrl), a silencing siR1 construct (siR1), a siR1 construct together with constitutively active PAK3 (siR1 + caPAK3), or a siR1 construct together with wild-type PAK3 (siR1 + wtPAK3). Note the reversal of the ARHGEF6 phenotype by caPAK3, but not wtPAK3. Bar, 3 µm. (B) Blind quantitative analysis of spine parameters show that the decrease in large mushroom-type spines induced by siR1 transfection (dashed bar) is reversed by co-transfection with constitutively active PAK3 (black bar), but not with wild-type PAK3 (grey bar). Data are mean ± s.e.m., n=8-20; *P<0.05). (C,D) Same as in B, but with regard to proportion of filopodia and elongated spines (>=1.8 µm; n=8-20; *P<0.05). (E) Cumulative distribution plot of the spine lengths and spine head diameters measured under control conditions (open circles), in siR1 transfected cells (filled circles), in cells cotransfected with siR1 and constitutively active PAK3 (open squares) and in cells co-transfected with siR1 and wild-type PAK3 (filled squares).

 

Figure 6
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  		Fig. 6. Effects of double knockdown and reverse rescue experiments between ARHGEF6 and PAK3. (A) The abnormal phenotype obtained in a cell co-transfected with EGFP, ARHGEF6 siR1 and PAK3 siRNA (left) and in a cell co-transfected with EGFP, PAK3 siRNA and wild-type ARHGEF6 (right). Bar, 2 µm. (B) Changes in large mushroom-type spines (spine head diameter >0.7 µm) observed in control, EGFP-transfected cells (white bar), in ARHGEF6 siRNA transfected cells (siGEF, dashed bar), in double knockdown cells transfected with EGFP, ARHGEF6 siR1 and PAK3 siRNA (siGEF + siPAK3, black bar), and in reversed rescue experiments (cells transfected with EGFP, PAK3 siRNA and wild-type ARHGEF6; siPAK3 + wtGEF, grey bar). Data are mean ± s.e.m. of 5-11 cells; *P<0.05. (C) Same as in B, but for filopodia. (D) Same as in B, but for elongated spines (>=1.8 µm).

 

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© The Company of Biologists Ltd 2006