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First published online 4 April 2006
doi: 10.1242/jcs.02896

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Temporally and spatially coordinated roles for Rho, Rac, Cdc42 and their effectors in growth cone guidance by a physiological electric field

School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland, AB25 2ZD, UK

View larger version (36K): [in a new window]   Fig. 1. Collective inhibition of Rac, Rho and Cdc42 with toxin B attenuates cathodal growth cone steering by an EF. (A) Mean angle turned by growth cones during 5 hours. In the absence of an EF, growth cones migrate randomly (mean angle turned would be 0° for random migration) but, in an EF of 150 mV/mm, growth cones turn towards the cathode. Negative values indicate net cathodal deflection. The number of growth cones measured is in parentheses. The mean angle turned at 5 hours was compared with `no drug + EF' using a Student's two-tailed t test; TxB, toxin B; ###P<0.0001. (B) Percentage of growth cones (see A for total) that turn towards the cathode (filled bars) or anode (open bars) in 5 hours. The dotted line is the expected frequency for random orientation (33%). Asterisks compare cathodal or anodal frequencies with no drug + EF; ***P<0.001. (C) Mean rate of growth cone advance during 5 hours of EF exposure compared with no drug + EF (black bar) using a two-tailed Student's t test. ##P=0.002; ###P<0.0001. See A for number of growth cones. (D) Composite drawings made from images of individual, dissociated neurons at the end of a 5-hour experiment. Somas were superimposed at the coloured dot and the path of each neurite was traced. The EF vector is horizontal, with cathode at left and anode at right. Bar, 100 µm for all drawings. ns, not significant.

View larger version (42K): [in a new window]   Fig. 2. Effect of selective inhibition of Rac, Rho or Cdc42 and elevation of Rho on EF growth cone guidance. See Fig. 1 for format. No drug + EF data are repeated from Fig. 1 for ease of comparison. (A) Mean angle turned since the start. #P<0.05; ##P<0.005; ###P<0.0001. (B) Frequency of cathodal (filled bars) and anodal (open bars) turning compared with no drug + EF (black bar). *P<0.05; **P=0.002; ***P<0.001. (C) The rate of neurite extension in an EF for 5 hours. #P<0.005; ##P=0.0035; ###P<0.0001. (D) Composite drawings of neurite paths for EF-treated cells. Compare with no drug in Fig. 1D. Bar, 100 µm for all drawings. ns, not significant.

View larger version (58K): [in a new window]   Fig. 3. Anodal RhoA elevation correlates spatially with collapsed morphology. (A) Mean anode-to-cathode ratios for Rho immunofluorescence intensity, number of filopodia and lamellipodial area for 40 growth cones (350 total filopodia) oriented within 45° of the EF direction and 13 growth cones with no EF (182 total filopodia). P values (p; two-tailed Student's t test) compare no EF and +EF ratios. A ratio of 1.0 (dotted line) indicates a symmetric growth cone, whereas ratios >1 and <1 indicate relative anodal and cathodal bias, respectively. (B,D) Confocal images of growth cones in100 nM LPA labelled with Rhodamine-phalloidin (red) and an antibody to RhoA (green). Image planes have been merged. (C,E) Confocal image of RhoA immunofluorescence with fluorescence intensity pseudocoloured on the scale shown. Dotted outlines indicate the regions used to calculate ratios. (F) RhoA immunofluorescence intensity plot for the cell in G. The green line represents Rho fluorescence measured along a line extending from the tip of the cathode-facing growth cone, along the neurite contour to the tip of the anode-facing growth cone. The black line represents fluorescence intensity when the same line is shifted to a background position near, but not overlapping, the cell. Asterisks indicate corresponding regions in image G. (G) A neuron in an EF for 5 hours labelled as in B. Insets (H,I,K,L) show detail of cathode-facing and anode-facing growth cones. (J,M) Fluorescence intensity plots for the lines indicated on I and L. Mean intensities (±s.e.m.) for the cathode-facing (blue) and anode-facing (red) sides of each plot are indicated by black bars. Ratios indicate mean anode intensity compared with mean cathode intensity for each growth cone. Cathode-versus anode-facing means were compared with a two-tailed Student's t test.

View larger version (39K): [in a new window]   Fig. 4. Inhibition of Rho effectors attenuates EF-induced growth cone guidance. Format is as in Fig. 1. (A) Mean angle turned during 5 hours. No drug + EF data are repeated from Fig. 1 for comparison; ns, not significant; #P<0.05; ##P=0.0002; ###P<0.0001. (B) Frequency of cathodal or anodal turning. *P<0.05; ***P<0.001. (C) Rate of growth cone advance in an EF. ##P=0.0002; ###P<0.0001. (D) Composite drawings of neurons in an EF (cathode to the left) for 5 hours. Compare with no drug + EF in Fig. 1D. Bar, 100 µm for all drawings.

View larger version (44K): [in a new window]   Fig. 5. Effects of inhibition of myosin-based contraction (using BDM or wortmannin) or PI 3-kinase (using LY294002 or wortmannin) on EF-induced growth cone guidance. Formatted as in Fig. 1. No drug + EF data are repeated from Fig. 1; ns, not significant compared with revelant no drug + EF or DMSO + EF control. (A) Mean angle turned. #P<0.05; ##P<0.0004; ###P<0.0001. (B) Frequency of turning during 5 hours. *P<0.05; ***P<0.001. (C) Growth rates during 5 hours of EF exposure. ##P=0.0002 compared with no drug + EF (black bar); P<0.0001 compared with DMSO + EF (grey bar). (D) Composite drawings of neurons in an EF (cathode to the left) for 5 hours. Bar, 100 µm for all drawings. Compare BDM and DMSO drawings to no drug + EF in Fig. 1D, and compare wortmannin and LY294002 to DMSO.

View larger version (41K): [in a new window]   Fig. 6. Effect of inhibition of MAPK signalling on EF-induced growth cone guidance. Format as is Fig. 1. Control data are repeated from Fig. 1; ns, not significant. (A) Mean angle turned during 5 hours is not affected by any inhibitor. (B) Percentage of growth cones that turn cathodally or anodally during 5 hours. None of the inhibitors affected the frequency of cathodal turning and only SB202190 slightly increased the anodal frequency compared with the DMSO control; *, P<0.05; ***P<0.0001. (C) Rate of growth cone advance during 5 hours in an EF. P values compared with no drug + EF: DMSO, ###P<0.0001; SB203580, #P<0.05. P values compared with DMSO + EF: U0126 and SB202190, ###P<0.001. (D) Composite drawings of neurons in an EF (cathode to the left) for 5 hours. Bar, 100 µm for all drawings. Compare U0126 and SB202091 to DMSO at top of panel and compare SB203580 to no drug + EF in Fig. 1D.

View larger version (44K): [in a new window]   Fig. 7. Working model for cathodal growth cone turning in an EF. See Discussion for detail. (A) The current model for growth cone steering of Xenopus growth cones by an EF. Pathway components implicated previously in cathodal turning are in boxes and grey ovals indicate components explored in the present study. Pharmacological inhibitors used in the present study are shown in grey text at their points of action. (B) Proposed pathway linking Rho GTPase activity to cathodal turning. In this scheme, Cdc42 and Rac signalling dominate cathodally, with concurrent suppression of Rho. Conversely, Rho signalling dominates anodally, with suppression of Cdc42/Rac activity through Rho activation (not shown). Thus, on the anode-facing side of the growth cone, the signalling events and consequences on cytoskeletal dynamics would be opposite to those indicated. Pathway components studied in this study are indicated in grey ovals and the inhibitors used (with the exception of LPA, which activates Rho) are indicated in grey text at their sites of action. Although the pathways are roughly linear for ease of presentation, extensive cross-talk would be expected.