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First published online February 22, 2006
doi: 10.1242/10.1242/jcs.02801

Journal of Cell Science 119, 951-957 (2006)
Published by The Company of Biologists 2006
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PTEN couples Sema3A signalling to growth cone collapse

Neil H. Chadborn1,*, Aminul I. Ahmed1,*, Mark R. Holt2, Rabinder Prinjha3, Graham A. Dunn2, Gareth E. Jones2 and Britta J. Eickholt1,{ddagger}

1 MRC Centre for Developmental Neurobiology, King's College London, Guy's Campus, London, SE1 1UL, UK
2 Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London, SE1 1UL, UK
3 GlaxoSmithKline, Neurodegeneration Research, Neurology and GI CEDD, Third Avenue, Harlow, Essex, CM19 5AW, UK


Figure 1
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  		Fig. 1. Inhibition of PI3K is sufficient to induce a growth cone collapse. (A) Single frames show a DRG growth cone before (0'), and following incubation with 10 µM LY294002 every 2 minutes (see Movie 1 in supplementary material). (B) Data charting the displacement (in µm + s.e.m.) of at least 15 growth cones in each of three independent experiments. LY294002 (10 µM) or control (DMSO) was applied to DRG cultures after 40 minutes (dotted line). Treatment with the inhibitor induces a retraction within 5 minutes and neurons start extending at half the original rate within 15 minutes. This response coincides with a drop in growth cone area, followed by the recovery in area during recovery of forward movement. (C) In the presence of the GSK-3 inhibitors, LiCl (20 mM), SB216763 (10 µM) or SB415286 (40 µM), the LY294002-induced growth cones collapse (10 µM, 10 minutes) is reduced. Each data point is the mean ± s.e.m. of six experiments, with n≥60 growth cones in each experiment. *P≤0.006; **P≤0.01. Scale bar, 10 µm.

 

Figure 2
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  		Fig. 2. Increases in PIP3 by activation of PI3K or inactivation of PTEN antagonises Sema3A-induced growth cone collapse. (A) Chick DRG explants were cultured overnight in the presence of NGF. Prior to application of Sema3A-Fc at 1 µg/ml, the cultures were incubated with the PI3K-activating 740Y-P peptide at 40 µg/ml for 30 minutes, with the 740Y-P peptide and LY294002 (10 µM), or with the Y-P-peptide 1309 (at 40 µg/ml). (B) GFP, GFP-PTEN or phosphatase-deficient GFP-PTEN C124S were nucleofected into chick DRG neurons, cultured for 24 hours and stimulated with Sema3A as before. Each data point is the mean ± s.e.m. of at least three independent experiments; *P<0.05.

 

Figure 3
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  		Fig. 3. PTEN is required for Sema3A-mediated Akt/GSK-3 signalling in N1E-115 cells. N1E-115 cells were transfected with GFP, GFP-PTEN or GFP-PTEN C124S (GFP-PTEN CS) and stimulated with Sema3A (1 µg/ml) for 5, 10, 20 or 30 minutes (A,B,C). Western blots using P(Ser473)-Akt and P(Ser9)GSK-3ß antibodies reveal a decrease in phosphorylation following Sema3A treatment in GFP- and GFP-PTEN-expressing cells, but not in cells expressing GFP-PTEN C124S. (D) N1E-115 cells were transfected with a nonspecific siRNA (*) or PTEN siRNA, and stimulated with Sema3A as previously. Knockdown of PTEN rendered NIE-115 cells insensitive to changes in GSK-3 and Akt phosphorylation in response to Sema3A. (E) In addition, the Sema3A response at 10 minutes (S) was compared with LY294002 treatments (LY, at 10 µM for 10 minutes). (F) Normalised relative band density (P-Akt/Akt, P-GSK-3/GSK-3) of each transfection experiment (mean + s.e.m. of three experiments).

 

Figure 4
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  		Fig. 4. PTEN is enriched in the axon of DRG neurons where it accumulates with microtubules. (A) A single confocal section taken through an E18 trunk of a rat embryo labelled with anti-ßIII-tubulin (left) and anti-PTEN (middle) antibodies. PTEN is present in the nucleus of DRG neurons (*) and the surrounding cytosol and in the axons of the peripheral projection of the DRGs (arrowhead). (B) Rat E18 DRG neurons were triple-labelled with anti-ßIII-tubulin (left), anti-PTEN (middle) antibodies and phalloidin (blue channel, right). In the growth cone, PTEN colocalises with the microtubule in the C-domain (arrow), whereas little signal is detected in the P-domain or the growth cone membrane (arrowhead). Bars, 10 µm.

 

Figure 5
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  		Fig. 5. PTEN and GFP-PTEN are enriched at microtubules in N1E-115 cells. (A) NIE-115 cells were labelled with anti-ßIII-tubulin (left) and anti-PTEN (middle) antibodies. Similarly to primary DRG neurons, there is an accumulation of endogenous PTEN with microtubules in N1E-115 cells. (B,C) NIE-115 cells were transfected with a control siRNA (B) or PTEN specific siRNA (C), and labelled with the anti-PTEN antibody (left) and Phalloidin to visualise F-actin. Confocal micrographs, taken with identical scanning parameters, demonstrate the loss of PTEN signal in the PTEN siRNA-treated cells. (D) Similarly to the distribution of endogenous PTEN, GFP-PTEN accumulates with microtubules in N1E-115 cells. Bars, 10 µm.

 

Figure 6
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  		Fig. 6. PTEN accumulates at the growth cone membrane during Sema3A mediated growth cone collapse. (A) DRG neurons from E18 rat embryos were nucleofected with GFP-PTEN and cultured overnight in vitro. Growth cones were imaged by confocal time-lapse microscopy at 1 frame every 30 seconds. The presented selection of images shows a growth cone 3 minutes before (-3', -2', -1') and during the response to Sema3A, which was applied at 0'. Following Sema3A application, fluorescent signals of GFP-PTEN increase uniformly at the membrane. See also Movie 2 in supplementary material. (B) A 3-pixel section in each frame was taken at the level indicated at 16' in A and pasted side by side. To facilitate the visualisation of movement and changes in relative fluorescence intensity at the membrane over time, we applied pseudo colour (see arrowhead). Following Sema3A addition at 0' (arrow) relative levels of PTEN increased at the membrane after 4 minutes (asterisk). Bar, 10 µm.

 

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