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First published online 17 July 2007
doi: 10.1242/jcs.006346

Journal of Cell Science 120, 2683-2693 (2007)
Published by The Company of Biologists 2007
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The neuronal Arf GAP centaurin {alpha}1 modulates dendritic differentiation

Carlene D. Moore1, Erin E. Thacker1,*, Jennifer Larimore1, David Gaston1, Alison Underwood1, Brian Kearns1,{ddagger}, Sean I. Patterson2, Trevor Jackson3, Chris Chapleau1, Lucas Pozzo-Miller1 and Anne Theibert1,§

1 Department of Neurobiology and Civitan International Research Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA
2 IHEM-CONICET, Departmento de Morfo-Fisiología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina
3 Departments of Physiology and Dermatology, School of Clinical and Laboratory Sciences, Medical School, University of Newcastle upon Tyne, NE2 4HH, UK


Figure 1
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  		Fig. 1. Developmental expression and localization of endogenous centaurin {alpha}1 in the rodent brain. (A) Immunoblot analysis of centaurin {alpha}1 protein from lysates (25 µg total lysate per lane) from whole brain at different developmental stages from E10 to P42 (=adult). (B) Immunoblot analysis of centaurin {alpha}1 and synaptic markers in fractionated P23 whole rat brain. H, whole brain homogenate; P2, crude microsomes and synaptosomes; S2, supernatant; Syn, purified synaptosomes; 20 µg of each fraction was loaded per lane. (C) Immunoblot analysis of centaurin {alpha}1 in the synaptosome fraction prepared from the developing brain. 10 µg of each fraction was loaded per lane. (D) Immunohistochemistry with anti-centaurin {alpha}1 antibody in hippocampus from P28 rat. DG, dentate gyrus.

 

Figure 2
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  		Fig. 2. Expression of endogenous centaurin {alpha}1 in primary cultured dissociated hippocampal neurons. (A) Immunoblot showing developmental expression of centaurin {alpha}1 and the specific synaptic proteins PDS95, spinophilin (Spino) and synaptotagmin (Syn) in primary cultured neurons at 3, 7, 14 and 21 DIV. 20 µg of total lysate was loaded per lane. (B) Indirect immunofluorescence shows endogenous centaurin {alpha}1 localization (red) and levels at 3, 7, 14 and 21 DIV. Bars, 10 µm.

 

Figure 3
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  		Fig. 3. Localization of endogenous centaurin {alpha}1 in primary cultured dissociated hippocampal neurons. Indirect immunofluorescence was used to visualize endogenous centaurin {alpha}1 localization (red) compared with microtubule associated protein 2 (MAP-2), tau, synaptophysin (syn), PSD-95 and spinophilin (Spin) in 14 DIV neurons. Arrows indicate areas of juxtaposition; arrowheads indicate areas of colocalization. Bars, 10 µm.

 

Figure 4
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  		Fig. 4. Effects of knockdown of centaurin {alpha}1 protein by siRNA in dissociated cultured hippocampal neurons. Hippocampal neurons were transfected after dissociation (0 DIV) and cultured for 3, 7 and 14 DIV. (A,B) Immunoblot analysis of endogenous centaurin {alpha}1 in hippocampal cultures. Total cell lysates (12.5 µg lysate per lane) were probed with anti-centaurin {alpha}1 and an anti-actin antibodies, which were used to assess actin levels for calculation of the knockdown percentage. (A) Lane 1, scrambled (Scr) siRNA 3 DIV; lane 2, rat centaurin {alpha}1 (rCena1) siRNA 3 DIV; lane 3, Scr siRNA 7 DIV; lane 4, rCena1 siRNA 7 DIV; lane 5, Scr siRNA 14 DIV; lane 6, rCena1 siRNA 14 DIV. (B) Lane 1, Scr siRNA; lane 2, rCena1 siRNA; lane 3, rCena1 siRNA plus human Flag-centaurin {alpha}1. (C) Indirect immunofluorescence showing endogenous centaurin {alpha}1 (red) compared with beta-tubulin (green) to visualize neuronal morphology at 3 and 7 DIV. Double-stranded scrambled RNA control is shown in left-hand panels and double-stranded siRNA to rat centaurin {alpha}1 in the right-hand panels. Bars, 20 µm. (D) Rescue by expression of human centaurin {alpha}1. Neurons were transfected with rat centaurin {alpha}1 siRNA, GFP and human centaurin {alpha}1. Indirect immunofluorescence showing endogenous plus heterologous centaurin {alpha}1-(red), beta-tubulin (blue) to visualize neuronal morphology and GFP (green) to label neurons co-expressing human Flag centaurin {alpha}1 at 3 and 7 DIV. Bars, 20 µm. (E) Quantification of the effects of siRNA knockdown of centaurin {alpha}1 at 3 and 7 DIV and rescue with human centaurin {alpha}1 compared with scrambled siRNA control. The data were quantified from three independent experiments (n=30 at 3 DIV, n=25 at 7 DIV cells for each condition). Data represent mean ± s.e. *P<0.05.

 

Figure 5
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  		Fig. 5. Effects of overexpression of wild-type centaurin {alpha}1 in developing dissociated hippocampal neurons. Hippocampal neurons were transfected by Nucleofection after dissociation (0 DIV) and cultured for 3, 7 and 14 DIV. (A) Fluorescence (GFP, green) and indirect immunofluorescence (beta-tubulin; red, 3 DIV; blue, 7 and 14 DIV; Flag-tagged centuarin {alpha}1, red) were used to visualize transfected neurons and neuronal morphology. GFP control (left panels). GFP plus Flag-centaurin {alpha}1 (right panels). Insets show higher magnification. Bars, 20 µm. (B) Quantification of the effects of expression of FLAG-centaurin {alpha}1 at 3 and 7 DIV compared with GFP control. The data was quantified from three independent experiments (n=50 cells from 3 DIV and n=30 cells from 7 DIV, respectively, for each condition). Data represent mean ± s.e. *P<0.05.

 

Figure 6
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  		Fig. 6. Effects of overexpression of a GAP-inactive mutant centaurin {alpha}1 on dendritic differentiation. Hippocampal neurons were transfected after dissociation (0 DIV) and cultured for 3-14 DIV. (A) Fluorescence (GFP, green) and indirect immunofluorescence (beta-tubulin, red) were used to visualize transfected neurons and neuronal morphology. Expression of GFP control (left panels) compared with GFP plus Flag-R49K centaurin {alpha}1 (right panel). Boxed areas show a higher magnification. Bars, 20 um. (B) Quantification of the effects of overexpression of Flag-R49Kcentaurin {alpha}1 for 3 and 7 DIV compared with GFP control. The data was quantified from three independent experiments (n=50 cells at 3 DIV and 30 cells at 7 DIV). Data represent mean ± s.e. *P<0.05.

 

Figure 7
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  		Fig. 7. Effects of overexpression of wild-type and GAP-inactive centaurin {alpha}1 on dendritic protrusions in dissociated cultured hippocampal neurons. Dissociated hippocampal neurons were transfected at 1 DIV and cultured for 13 DIV. (A) Fluorescence (GFP, green) and indirect immunofluorescence (spinophilin or neurabin, red) were used to visualize transfected neurons and morphological effects. Expression of GFP control (left panels) compared with GFP plus Flag-centaurin {alpha}1 (middle panel) and GFP plus Flag-R49Kcentaurin {alpha}1 (right panel). Bars, 10 µm. (B) Quantification of the number of dendritic protrusions per 10 µm dendrite length in neurons expressing GFP, GFP plus Flag-centaurin {alpha}1 and GFP plus Flag-R49K centaurin {alpha}1. Data represent mean ± s.e. *P<0.05.

 

Figure 8
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  		Fig. 8. Centaurin {alpha}1 localizes to dendritic spines and regulates dendritic spine density in CA1 neurons in organotypic hippocampal slice cultures. (A) Representative dendritic segments from CA1 pyramidal cells in organotypic hippocampal slice cultures transfected with eYFP and Flag-centaurin {alpha}1. Centaurin {alpha}1 was visible in the dendritic shaft and spines (arrows). Bar, 25 µm. (B) Representative dendritic segments of CA1 pyramidal neurons from organotypic hippocampal slice cultures transfected with eYFP and either dsRed, Flag-centaurin {alpha}1 or Flag-R49K centaurin {alpha}1. Bars, 2 µm. (C) Quantification of spine density in each condition. Data represent mean ± s.e. *P<0.00016.

 

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© The Company of Biologists Ltd 2007