QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 24 July 2008
doi: 10.1242/dev.024133

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Moore, S. W. Articles by Kennedy, T. E. Search for Related Content PubMed PubMed Citation Articles by Moore, S. W. Articles by Kennedy, T. E. Social Bookmarking                         What's this?

Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1

Centre for Neuronal Survival, Montreal Neurological Institute, Program in NeuroEngineering, Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec H3A 2B4, Canada.

View larger version (41K): [in this window] [in a new window]   Fig. 1. Dorsal spinal cord neurons express RhoA, ROCK and PRK family members. (A) RT-PCR amplification indicates that RhoA, RhoB and RhoC, as well as ROCKI, ROCKII, PRK1 and PRK2 are expressed by SCNs. (B) Western blot analysis of SCN lysate using RhoA, ROCKII and PRK2 antibodies. Immunofluorescent localization of RhoA (C,D), ROCKII (G,H) and PRK2 (K,L). In E,F,I,J,M,N. DCC is labeled green and nuclei blue; RhoA (E,F), ROCKII (I,J) and PRK2 (M,N) are labeled in red. Scale bar: 20 µm for C,E,G,I,K,M; 10 µm for D,F,H,J,L,N.

View larger version (18K): [in this window] [in a new window]   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.

View larger version (40K): [in this window] [in a new window]   Fig. 3. Inhibiting RhoA signaling increases DCC-dependent SCN axon outgrowth evoked by netrin 1. E13 rat dorsal spinal cord explants were cultured for 14 hours with various amounts of netrin 1 in the presence of 10 µg/ml C3-07 or 10 µM Y27632. (A-I) SCN axons were labeled using the 4D7 monoclonal antibody against Tag1. (J) C3-07 increased total outgrowth per explant by 155% (n=5, P=0.006) at 50 ng/ml netrin 1, 66% (n=4, P=0.041) at 200 ng/ml netrin 1 and 126% (n=4, P=0.003) at 600 ng/ml netrin 1. Y27632 increased outgrowth by 404% (n=5, P<0.001) at 50 ng/ml netrin 1, 137% (n=5, P<0.001) at 200 ng/ml netrin 1 and 236% (n=5, P=0.000) at 600 ng/ml netrin 1 (J). (K) DCC-fb antibody (5 µg/ml) significantly reduced axon outgrowth in the presence of C3-07 or Y27632 at each netrin 1 concentration (n=5, P<0.01). Specifically, total outgrowth with C3-07 was reduced by 74% (n=5, P=0.001) at 50 ng/ml netrin 1, by 70% (n=5, P<0.001) at 200 ng/ml netrin 1 and 79% (n=5, P<0.001) at 600 ng/ml. Total outgrowth with Y27632 was reduced by 57% (n=5, P<0.001) at 50 ng/ml netrin 1, 55% (n=5, P<0.001) at 200 ng/ml netrin 1 and 93% (n=5, P<0.001) at 600 ng/ml netrin 1. *P<0.05, **P<0.01. Tukey post-hoc tests of means. Error bars indicate s.e.m. Scale bar: 100 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Increased RhoA activity reduces SCN axon outgrowth evoked by netrin 1. (A-I) SCN explants expressing RFP, wt-RhoA or ca-RhoA were cultured for 14 hours in the presence of 200 ng/ml netrin 1. (B) Wt-RhoA reduced total outgrowth by 53% (n=21, P<0.001) and (C) ca-RhoA reduced it by 79% (n=23, P<0.001) compared with RFP infected explants (A). C3-07 increased RFP infected explant outgrowth by 32% (n=23, P<0.001) (D), wt-RhoA by 72% (n=20, P<0.001) (E) and ca-RhoA by 122% (n=20, P<0.001) (F). Y27632 increased RFP infected explant outgrowth by 53% (n=19, P<0.001) (G), wt-RhoA by 155% (n=20, P<0.001) (H) and ca-RhoA by 288% (n=22, P<0.001) (I). (J)**P<0.001 compared with control RFP explants. ##P<0.001 compared with wild type or CA explants. Tukey post-hoc tests of means. Error bars indicate s.e.m. Scale bar: 100 µm.

View larger version (95K): [in this window] [in a new window]   Fig. 5. Inhibiting RhoA signaling promotes SCN axon turning to netrin 1. (A,B) Aggregates of netrin 1-expressing HEK 293-EBNA cells were cultured alongside explants of E11 rat spinal cord and deflection distances measured as shown (A), and graphed (B) as the percent distance relative to the absence of drugs. (C-H) Explants were cultured for 40 hours in the absence of RhoA inhibitors (C,D), or in the presence of 10 µg/ml C3-07 (E,F) or 10 µM Y-27632 (G,H). SCN axons were fluorescently labeled for Tag1 (4D7). Both C3-07 (E, n=30, P=0.005) and Y27632 (G, n=30, P=0.007) resulted in a 16% increase in the turning distance. DCC function blocking antibodies (5 µg/ml) reduced turning in the absence of drug (D, n=24, P=0.001), as well as turning in the presence of 10 µg/ml C3-07 (F, n=21, P<0.001) or 10 µM Y27632 (H, n=24, P<0.001). Tukey post-hoc tests of means. Error bars indicate s.e.m. **P<0.001 compared with control explants without Y27632, C3-07 or DCC-fb. Scale bar: 20 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 6. Inhibiting RhoA signaling increases the amount of plasma membrane DCC in SCNs. Plasma membrane levels of the netrin 1 receptor DCC in SCN in vitro were evaluated using biotinylation (A) or immunofluorescence (B-H). Cell-surface levels were measured following 1-hour incubations with either 10 µg/ml C3-07 (C3) or 10 µM Y27632 (Y) and 5 minutes following application of 50 ng/ml netrin 1 (Net), as indicated. In the biotinylation assay (A), C3-07 caused a 50% (n=8, P=0.002) and Y27632 a 78% (n=8, P<0.001) increase in the amount of DCC in the absence of netrin 1. Netrin produced a 37% (n=8, P=0.021) increase, whereas the combination of both netrin and C3-07 or Y27632 generated a 59% (n=8, P<0.001) or 64% (n=8, P<0.001) increase, respectively. No significant change in the amount of cell-surface Tag1 was detected. (B) The ratio plasma membrane DCC immunofluorescence to total cellular DCC immunofluorescence within the growth cone increased in the presence of C3-07 by 9.9% (n=85, P=0.006), Y27632 by 8.7% (n=87, P=0.022), netrin 1 by 6.3% (n=124, P=0.048), netrin 1/C3-07 by 11.0% (n=80, P=0.002) and netrin 1/Y-27632 by 10.7% (n=90, P=0.001). Grayscale images of the plasma membrane (PM) and total DCC immunofluorescence in C-H were converted to heatmaps using the `Fire' lookup table of Image J (NIH, Bethesda, MD). *P<0.05, **P<0.01. Tukey post-hoc tests of means. Error bars indicate s.e.m. Scale bar: 10 µm.

View larger version (45K): [in this window] [in a new window]   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).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter