Multidimensional analysis of small GTPases as key molecules in the neural network formation by using activity-imaging

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In this study, we performed the activity-imaging of Ras and Rho GTPases using GFP-based FRET probes in PC12 Cells, DRG neurons, and hippocampal neurons.1.During NGF-induced neurite outgrowth in PC12 cells, the activity-imaging and RNA interference study showed that local PIP3 accumulation after NGF stimulation recruited Vav2 and Vav3 to the plasma membrane and thereby activated Rac1 and Cdc42 ; this Vav2/3-dependent activation of Rac1 and Cdc42 promoted the formation of short processes leading to neurite outgrowth. We also provide evidence that Vav2 and Vav3 are involved in the local activation of PI3-kinase at the protrusions after NGF stimulation. Therefore, this study demonstrates that Vav proteins are constituents for the first time that Vav proteins are constituents of the PIP3-dependent positive feedback loop that cycles locally with morphological changes.2.We visualized RhoA/Rac1/Cdc42 activities during laminin-induced growth cone advance of DRG neurons and N1E-115 cells. The Rac1 and Cdc42 activities were high in the P-domain of growth cones. Against a model involving RhoA down-regulation at the periphery of protruding growth cones, we found that the RhoA activity was higher in the P-domain than in the C-domain and axon shaft, and that a high level of RhoA activity was maintained in the extending part of growth cones. In LPA-treated N1E-115 cells, well-developed neurites with growth cones showed RhoA activation, but sustained their extended morphology until they were drawn toward the contracting somata. Thus, RhoA activation in the shaft results in neurite retraction, whereas high RhoA activity in the P-domain is necessary to retain the spread morphology of nerve growth cone.3.We visualized the activities of Ras and Rho GTPases within the postsynaptic sites using high-density cultures of hippocampal neurons. This study showed that each member of these GTPases had a peculiar pattern of activation in the postsynaptic sites.