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First published online 8 May 2007
doi: 10.1242/jcs.000703

Journal of Cell Science 120, 1859-1867 (2007)
Published by The Company of Biologists 2007
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The Down syndrome critical region protein TTC3 inhibits neuronal differentiation via RhoA and Citron kinase

Gaia Berto1, Paola Camera1, Carlo Fusco2, Sara Imarisio3, Chiara Ambrogio4, Roberto Chiarle4, Lorenzo Silengo1 and Ferdinando Di Cunto1,*

1 Molecular Biotechnology Center, Department of Genetics, Biology and Biochemistry, University of Turin, Italy
2 Division of Experimental Pathology, Institute of Pathology, University of Lausanne, Lausanne, Switzerland
3 Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrooke's Hospital, Cambridge, UK
4 Center for Experimental Research and Medical Studies and Department of Biomedical Sciences and Human Oncology, University of Turin, Italy


Figure 1
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  		Fig. 1. Interaction between Citron proteins and TTC3. (A) Domain structure of Citron proteins, TTC3 and the described mutants. Kinase domain (black), Rho-binding domain (black-box), coiled coil region (grey), C1 domain (cyan), PH domain (pink), CNH domain (red), Proline-rich region (orange), PDZ-binding domain (brown), TPR motifs (yellow), Citron binding region CBR (green) and ring finger domain (blue) are indicated. The sequences used as bait, and found as prey are indicated. (B) Total cell lysates from HEK293 cells co-transfected with GFP-CIT-N and Myc-CBR were immunoprecipitated with anti-Citron antibodies and analyzed by western blotting as indicated. (C) Total cell lysates from HEK293 cells co-transfected with GFP-TTC3 and CIT-N or CIT-K were immunoprecipitated with anti-Citron antibodies and analyzed by western blotting as indicated. (D) PC12 cells were co-transfected with GFP-TTC3 and allowed to differentiate by NGF treatment for 2 days after transfection. Total cell lysates were then immunoprecipitated with anti-GFP antibodies and endogenous CIT-K was revealed by western blotting. (E) The TTC3 mutants {Delta}Cterm and {Delta}CBR were co-transfected with CIT-K and analyzed as in C. (F) Myc-CIT-K was co-transfected in HEK293 cells with GFP-TTC3 (upper panel) or with GFP-{Delta}CBR (lower panel). Bar, 10 µm.

 

Figure 2
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  		Fig. 2. Inhibition of neurite extension by TTC3 overexpression. PC12 cells were transfected with GFP-empty, GFP-TTC3 and GFP-TTC3 mutants. The cells were treated with NGF 12 hours after transfection, allowed to differentiate for 3 days and analyzed by immunofluorescence microscopy. (A) Cells transfected with TTC3 showed a dramatic reduction of neurite sprouting, when compared to control cells. PHD, phalloidin; HOE, Hoechst 33258. Bar, 10 µm. (B) The percentage of differentiated cells, defined as those bearing at least one neurite longer than the main cell body axis, was quantitatively analyzed in the indicated transfections. The histograms represent the average of three independent experiments. All the differences were statistically significant (P<0.001, {chi}-square test), except the one detected between the {Delta}Cterm and {Delta}CBR mutants. Error bars represent s.d.

 

Figure 3
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  		Fig. 3. Knockdown of TTC3 in differentiated PC12 cells. (A) HEK293 cells were co-transfected with GFP-TTC3 and unrelated pDECAP control (pD) or pDECAP-TTC3 (pDT) plasmids. The expression of GFP-TTC3 and that of an internal loading control (Vinculin) were then evaluated by western blot 48 hours after transfection. (B) HEK293 cells were co-transfected with GFP-TTC3 and pCMV-GIN-ZEO sh-mir plasmids, expressing a double mismatch (pC) and two perfect match (pC-1 and pC-2) interfering RNAs. The reduction of GFP-TTC3 was evaluated as in A. (C) PC12 cells were transfected with the indicated control and RNAi plasmids. The levels of endogenous TTC3 mRNA were evaluated 48 hours after transfection by semi-quantitative RT-PCR, using internal control beta-actin primers. (D,E) PC12 cells were transfected with the indicated control and RNAi plasmids and treated with NGF for 3 days. Cells were then fixed, stained with phalloidin and analyzed by IF. The ratio between length of the main neurite and principal diameter of cell body was determined for at least 200 cells. Histograms represent the average of three independent experiments (D). The differences between control (gray bars) and RNAi (black bars) samples were statistically significant, as determined by the Student's t-test (P<0.0001). Error bars represent s.d. Bar, 10 µm.

 

Figure 4
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  		Fig. 4. Epistatic analysis of TTC3 and CIT-K in differentiating PC12 cells. (A) PC12 cells were co-transfected with GFP-TTC3 and pDECAP control (pD-CTRL) or pDECAP CIT-K (pD-CIT), allowed to differentiate for 3 days and analyzed as in Figs 2 and 3. The image shown in the lower panel corresponds to an exemplar binucleated cells, bearing long neurites. Bar, 10 µm. (B) Quantitative analysis of differentiated cells in the experiment described in A. The first bar represents the result of a parallel control sample co-transfected with a GFP empty vector and pD-CIT. (C) PC12 cells were co-transfected with the indicated combinations of Myc-CIT-K, unrelated Myc expression plasmid (Myc-CTRL), pD-CTRL and pDECAP-TTC3 (pD-TTC3). Slides were analyzed as in A and B, with the exception that transfected cells were identified by staining with anti-Myc antibodies. Error bars represent s.d.

 

Figure 5
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  		Fig. 5. Rho kinase inhibition is unable to revert the TTC3 overexpression phenotype in differentiated PC12 cells. (A) PC12 cells were transfected with an empty GFP or GFP-TTC3 construct, treated with NGF after 12 hours and allowed to differentiate for 2 days. Afterwards, the culture medium was changed and vehicle alone (MOCK) or Y27632 were added. The cells were kept in culture for additional 18 hours and then processed for immunofluorescence with anti GFP antibodies (green) and phalloidin (red). Bar, 10 µm. (B) Quantitative analysis of the percentage of differentiated cells (left panel) and of neurite length (right panel) in the above experiments, performed as in Figs 2 and 3, respectively.

 

Figure 6
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  		Fig. 6. Rho inhibition reverts the phenotype induced by TTC3 overexpression in PC12 cells. (A) PC12 cells were transfected with empty GFP or GFP-TTC3 constructs, either alone or in combination with HA-C3 expression plasmid. Cultures were treated with NGF after 12 hours, allowed to differentiate for 3 days and processed for IF. (B) Quantitative analysis of differentiated cells in the above experiment, performed as in Fig. 2. (C) HEK293 cells (upper panel) or PC12 cells (lower panel) were transfected with empty GFP or GFP-TTC3 expression vectors and analyzed after 48 hours by Rho pull-down assay, using recombinant Rhotekin Rho-binding domain (GST-RBD).

 

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© The Company of Biologists Ltd 2007