Fig. 1.

Inhibition of NHE1 activity reduces the activation of Rho GTPases by netrin-1 in growth cones. a. Netrin-1 activates Cdc42 and Rac1 and down-regulates RhoA in total cell lysates of mouse neocortical neurons. Neurons were treated with 100 ng/ml netrin-1 for 10 min. GST-PAK-CRIB and GST-rhotekin-RBD fusion proteins were used to pull-down activated Rho GTPases (which were then identified by their respective antibodies). RhoA-GTP/total RhoA ratio, as determined by gel module of Image J analysis software, decreased to 0.07 from 0.7 after netrin treatment. b. Acceptor photobleaching FRET of Rho GTPase activities in mouse neocortical neuron growth cones transfected with YFP-GTPase/Effector-binding domain-CFP fusion probes (i.e. Raichu probes). FRET efficiencies (E%) were quantified on a pixel-by-pixel basis. Compared to growth cones of WT neurons, Rho GTPase activities are reduced in growth cones of NHE1-null neurons-. *P < 0.05, **P < 0.01. Numbers of growth cones analyzed under each condition are shown in columns. c. Left panel: Quantification of FRET efficiencies (E%) in WT neuronal growth cones, in the absence or presence of netrin-1 (100 ng/ml for 10 min). Netrin-1 increased Rho GTPase activities above control values that in turn were abolished by cariporide (1 μM). *P < 0.05, **P < 0.01, ***P < 0.001. Numbers of growth cones analyzed under each condition are shown in the columns. Right panel: Representative pre- and post-bleach images of Rac1 FRET under the conditions indicated