Combined analysis of feedback loops consisting of G proteins and phospholipid in the formation of neural cirnuitry

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Neurite outgrowth upon extracellular cues is an essential event in early neuronal differentiation. Rac1 and Cdc42, members of Rho family GTPases, are involved in this process as regulators of actin cytoskeletal reorganization. I previously showed that NGF/TrkA. Signaling drives the cycling of a positive feedback loop comprised of PI3-kinase, Vav2/Vav3, Rac1/Cdc42, and actin cytoskeleton at neurite tips of PC12 cells using FRET imaging and RNAi techniques. In this study, I have demonstrated that SHIP2 is a critical component of the negative feedback on PIP_3 in NGF-induced neurite outgrowth. This negative feedback loop has been unexpected previously. Acute inhibition of TrkA transiently decreased Rac1/Cdc42 activity and the PIP_3 level to below basal levels, indicating the presence of NGF-dependent negative regulation. With the help of in Silico simulation, I speculated that the activation of PI-5-phosphatase for PIP_3 was involved. In agreement with this model, depletion of SHIP2 by RN … More A interference attenuated the inhibitor-induced super-suppression of Rac1/Cdc42 activity and the level of PIP_3. The critical role of SHIP2 in neurite outgrowth of PC12 cells was further supported by the finding that depletion of SHIP2 and PTEN, phosphatases for PIP_3, markedly potentiated NGF-induced Rac1/Cdc42 activation and PIP_3 accumulation, and significantly increased the number and the length of neurites. This increase in neurite number is possibly due to that the depletion of SHIP2 and PTEN disturbs the proper localization of PIP_3 and Rac1/Cdc42 activities. Further refinement of the computational model predicted a negative feedback from Rac1 to SHIP2, which was validated experimentally. I propose that the SHIP2-mediated negative feedback on PIP_3 coordinately works with the PI3-kinase-mediated positive feedback to form an initial protrusive pattern determined by Turing's reaction-diffusion system, and later, to punctuate the PIP_3 accumulation to maintain proper neurite outgrowth. This study emphasizes a useful intertwining of FRET imaging and system biology. Less