Figure 2. MET signaling activates the small GTPase Cdc42, which mediates its effects on dendritic and spine morphogenesis.

(a) Schematic illustration of an ex vivo prefrontal brain slice preparation (coronal sections from P9 mice) used for HGF treatment to initiate MET signaling. (b) MET activation, using p-MET (Y1234/1235) level as a surrogate, was induced by HGF, and blocked by MET kinase activity inhibitor PHA665752 (200 nM) (**p < 0.01. ND, not detected). (c) The PI3K inhibitor, Wortmanin (100 nM), did not affect p-MET levels, indicating PI3K activity was not required MET activation. (d) In both P9-10 ex vivo slices and DIV11 cortical neuron culture preparations, HGF (50 ng/ml) induces Cdc42 activation, measured by PAK P21 domain pull-down of the GTP-bound Cdc42. In contrast, no change of overall Cdc42 level was observed (*p < 0.05, **p < 0.01). (e) Cdc42 activation was blocked by either PHA665752 or Wortmanin, indicating it is dependent on MET kinase activity and downstream to PI3K activation (**p < 0.01). (f) Neuronal morphometric measurement using TDBTN and TDBL, and representative images of dendritic and spine morphology from cultured neurons transfected with Met cDNA, or in combination with Cdc42 loss-of -function plasmids (DN-Cdc42 or Cdc42 RNAi). (g) TDBTN quantification (normalized to GFP control neurons) reveals MET OE significantly increased TDBTN, which is significantly blocked by either DN-Cdc42 or Cdc42 RNAi co-transfection. Note Cdc42 RNAi alone significantly reduced TDBTN (*p < 0.05, **p < 0.01 compared with GFP, ns, not significant. ^##p < 0.01. Number of neurons quantified listed on bar). (h) MET OE increases TDBL, an effect that is also blocked by co-transfection with DN-Cdc42 or Cdc42 RNAi (*p < 0.05, **p < 0.01, ^#p < 0.05, ^##p < 0.01). (i) Enhanced MET signaling increases dendritic spine density, which is blocked by Cdc42 antagonism (**p < 0.01, ^##p < 0.01, ns, not significant).