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First published online 4 April 2006
doi: 10.1242/jcs.02897

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Growth cone steering by a physiological electric field requires dynamic microtubules, microfilaments and Rac-mediated filopodial asymmetry

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

View larger version (31K): [in a new window]   Fig. 1. Actin microfilaments are required for growth cone turning towards the cathode. (A) Mean angle turned in 3 hours; lat, latrunculin; n, number of growth cones measured; ns, not significant. *P<0.05; **P<0.005; ***P<0.0001. Black asterisks indicate comparison with `DMSO + EF' and grey asterisks indicate comparison with `DMSO, no EF'. (B) Mean migration rates for the growth cones in A. (C) The frequency of turning towards the cathode (filled bars) and the anode (open bars) for the same population. #P<0.002; ##P<0.001 compared with DMSO + EF. (D) Composite drawings of individual neurons at 3 hours made by superimposing cell bodies at the coloured dot and tracing individual neurite paths. For EF-treated cells, the EF vector is horizontal with the cathode to the left and the anode to the right. Bar, 100 µm, applies to all.

View larger version (86K): [in a new window]   Fig. 2. Actin and microtubule staining of growth cones exposed to an EF of 150 mV/mm (cathode to the left) in the presence of cytoskeleton inhibitors. Confocal images of Rhodamine-phalloidin (red) staining of F-actin and -tubulin (green). (A) No drug + EF. (B,C,F) Taxol (tax) + EF. Arrow in C indicates looped microtubules in the central region of the growth cone, enlarged in the inset. Arrow in F indicates the swelling that sometimes forms in neurons exposed to >20 nM taxol. It marks the point on the neurite where the growth cone was at the time of drug addition. (D,E) Latrunculin A (lat) + EF. Inset in D shows detail of the growth cone in the white rectangle. Actin staining in 25 nM latrunculin is punctate, indicated by arrow in E. (F) Taxol + EF. (G) Vinblastine + EF, Bars, 10 µm.

View larger version (39K): [in a new window]   Fig. 3. Microtubules are required for cathodal turning. (A) Mean angle turned in 3 hours; n, number of growth cones; noc, nocodazole; tax, taxol; vin, vinblastine. *P<0.05; **P<0.005; ***P<0.0001. Black asterisks indicate comparison with `DMSO + EF' (for noc and tax) or `no drug + EF' (for vin), and grey asterisks indicate comparison with `DMSO no EF' control or `no drug, no EF' control. (B) Mean rate of growth cone advance. (C) Frequency of turning towards the cathode (filled bars) or anode (open bars) during 3 hours. #P<0.002; ##P<0.001. (D) Composite drawings made from images of individual neurons at the end of 3 hours. Cell bodies have been superimposed and individual neurite paths traced. Bar, 100 µm, applies to all.

View larger version (41K): [in a new window]   Fig. 4. Dynamic analysis of growth cones in the presence and absence of an EF and inhibitors of the Rac and Cdc42 CRIB domains. (A) The mean angle turned at intervals of 10 minutes; n, number of growth cones. The angle turned in an EF (black spots) is no different than the no EF control (white spots) at 10 minutes; however, by 20 minutes, the angle turned is significantly more cathodal (P=0.0243) and becomes increasingly cathodal with time (P<0.005 from 30-50 minutes, and P<0.0001 from 60-180 minutes). The Rac (red spots) and Cdc42 (blue spots) peptides abolish turning for the entire 3 hours (P>0.05 throughout). (B) Frequency of growth cones that turn >15° towards the cathode. Symbols and number of growth cones are as for A. (C) The percentage of filopodia facing the cathode. The broken line indicates the expected frequency for randomly directed filopodia (50%); n, number of growth cones measured. (D) The percentage of total lamellipodial area facing the cathode. The dotted line represents the theoretical frequency for randomly directed lamellipodia (50%); n, number of growth cones.

View larger version (57K): [in a new window]   Fig. 5. Effects of inhibition of Rac or Cdc42 CRIB activity on cytoskeletal morphology. All growth cones were exposed to an EF (cathode at left) for 5 hours. (A) No drug + EF. (B-D) Growth cones treated with Rac 17-32 (rac). Note that microtubules (first column) appear less adherent to each other along their lengths. Numerous, uniformly distributed filopodia are present but lamellipodia are not well formed. (E,F) Growth cones in Cdc42 17-32 peptide (cdc). Microtubules (first column) appear loosely adherent to each other and they splay into the peripheral region of the growth cone more than when treated with Rac 17-32. Filamentous actin staining (second column) reveals large, well-spread lamellipodia and relatively fewer filopodia than when treated with Rac 17-32. When they are present, filopodia face the cathode. Bars, 10 µm.

View larger version (60K): [in a new window]   Fig. 6. Hypothetical mechanism by which Rho GTPases mediate growth cone steering towards the cathode. The concentrations of active Rac and Cdc42 (red) are relatively high on the cathode-facing side of the growth cone, but Rho activity (green) is relatively low on that side. Conversely, active Rho is relatively high anodally and active Rac and Cdc42 are low. Some candidate Rac and Cdc42 effectors are suggested. Others effectors, whose involvement has been tested, are presented in the companion paper in this issue (Rajnicek et al., 2006).