Journal: Development (Cambridge, England)

Article Title: Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1.

doi: 10.1242/dev.024133

Figure Lengend Snippet: Fig. 2. Netrin 1 inhibits RhoA in SCNs. G-LISA (A) and GST-RBD pulldown (B) assays were used to evaluate the relative amounts of RhoA-GTP in SCNs. GST-RBD pulldown assays measured a 27% (P=0.040) average reduction across three separate experiments. G-LISA assays measured an average reduction of 13% after 15 minutes of 200 ng/ml netrin 1 (n=23, P=0.002). This was blocked with either 5 μg/ml of DCC function blocking antibodies (DCC-fb) or 2 μg/ml of the extracellular domain of DCC (DCC-fc). Tukey post-hoc tests of means. Error bars indicate s.e.m.

Article Snippet: *Author for correspondence (e-mail: timothy.kennedy@mcgill.ca) Accepted 28 June 2008 D E V E LO P M E N T 2856 Tag1 (TG3) for western blot analysis (provided by Dr Thomas Jessell, Columbia University, New York, NY); rabbit anti-integrin β1 (AB1952, Chemicon, Temecula, CA); mouse IgG anti-RhoA (26C4) and goat antiDCC (A-20, Santa Cruz Biotechnology, Santa Cruz, CA); mouse IgG antiDCC (AF5) and Y27632 (Calbiochem, LaJolla, CA); DCC-fc, a protein chimera composed of the extracellular domain of mouse DCC and the Fc portion of human IgG1 (R & D Systems, Minneapolis, MN); mouse IgG anti-ROCKII (BD Biosciences, Mississauga, Canada); rabbit anti-PRK2 (Cell Signaling, Danvers, MA); DNase, poly-D-lysine (PDL, 70-150 kDa) and Hoechst 33258 (Sigma-Aldrich, Mississauga, Canada); and Neurobasal, FBS, B-27 supplement, GlutaMAX-1, penicillin-streptomycin, Ca2+/Mg2+free HBSS, Alexa 546-coupled phalloidin and Jasplakinolide were purchased from Invitrogen Canada (Invitrogen Canada, Burlington, ON).

Techniques: Blocking Assay

Journal: Development (Cambridge, England)

Article Title: Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1.

doi: 10.1242/dev.024133

Figure Lengend Snippet: Fig. 7. Inhibiting RhoA signaling promotes adhesion and growth cone expansion in response to netrin 1. (A) SCN adherence to netrin 1 is increased by 79% (n=12, P<0.001) in the presence of C3-07 (C3) or by 152% (n=12, P<0.001) with Y-27632 (Y). This adhesion was reduced (n=12, P<0.001) upon pre-incubation with either: netrin function blocking antibodies (anti-netrin) or by competition with the extracellular domain of DCC (DCC-fc receptor-body). Stabilization of filamentous actin with jasplakinolide (Jasp.) disrupted adhesion to netrin 1 and abolished the increased adhesion evoked by C3-07 or Y-27632. (B-D) Representative images of cells binding to netrin 1 substrates in the absence (B) or in the presence of C3-07 (C) or Y-27632 (D). (E-H) The expansion of SCN growth cones as they encounter a boundary of immobilized netrin 1 is consistent with netrin 1 functioning as an adhesive cue. (I-N) SCNs labeled with phalloidin (green), β-tubulin (red) and Hoechst (blue). The growth cones of SCNs grown on glass coverslips coated with PDL (I-K) are smaller than those grown on this same substrate with an additional coating of netrin 1 (L-N). (O) In the absence of netrin 1, the average growth cone increased by 67% (n=21, P=0.008) when treated with C3-07 and by 79% (n=20, P<0.001) when treated with Y27632. On a substrate of netrin 1, average growth cone area increased by 94% in the absence of inhibitors. A netrin 1 substrate also increased the average area of growth cones in the presence of C3-07 by 76% (n=22, P=0.006) and Y27632 by 70% (n=21, P=0.033). SCN were infected with herpes simplex viral vectors encoding with RFP (P), wt-RhoA (Q) or ca-RhoA (R). Neurons were visualized with Hoechst (blue), phalloidin (green) and with antibodies against myc (red) or endogenous RFP fluorescence (red). Growth cones area (S) was reduced by 61% when RhoA was overexpressed (n=37, P<0.001) or by 90% upon expression of ca-RhoA (n=20, P<0.001). (T) Working model illustrating the hypothesis that RhoA inhibition by netrin 1 enhances chemoattraction by facilitating DCC function, in part by recruiting additional DCC to the plasma membrane and by promoting DCC signaling mechanisms, such as increasing adhesion to immobilized netrin 1, that lead to membrane extension. Tukey post-hoc tests of means. Error bars indicate s.e.m. Scale bars: 200 μm in B-D; 20 μm in E-H; 10 μm in I-R. (A,O,S) *P<0.05; **P<0.01. (O) For the fifth and eighth bars, **P<0.01 relative to the second bar. For the third, sixth and ninth bars, *P<0.05 and **P<0.01 relative to the second, fifth and eighth bars, respectively (i.e. compared with the absence of DCC-fc).

Article Snippet: *Author for correspondence (e-mail: timothy.kennedy@mcgill.ca) Accepted 28 June 2008 D E V E LO P M E N T 2856 Tag1 (TG3) for western blot analysis (provided by Dr Thomas Jessell, Columbia University, New York, NY); rabbit anti-integrin β1 (AB1952, Chemicon, Temecula, CA); mouse IgG anti-RhoA (26C4) and goat antiDCC (A-20, Santa Cruz Biotechnology, Santa Cruz, CA); mouse IgG antiDCC (AF5) and Y27632 (Calbiochem, LaJolla, CA); DCC-fc, a protein chimera composed of the extracellular domain of mouse DCC and the Fc portion of human IgG1 (R & D Systems, Minneapolis, MN); mouse IgG anti-ROCKII (BD Biosciences, Mississauga, Canada); rabbit anti-PRK2 (Cell Signaling, Danvers, MA); DNase, poly-D-lysine (PDL, 70-150 kDa) and Hoechst 33258 (Sigma-Aldrich, Mississauga, Canada); and Neurobasal, FBS, B-27 supplement, GlutaMAX-1, penicillin-streptomycin, Ca2+/Mg2+free HBSS, Alexa 546-coupled phalloidin and Jasplakinolide were purchased from Invitrogen Canada (Invitrogen Canada, Burlington, ON).

Techniques: Incubation, Blocking Assay, Binding Assay, Adhesive, Labeling, Infection, Fluorescence, Expressing, Inhibition, Clinical Proteomics, Membrane