Edgeworth, Julie Ann; (2003) The role of Cdc42 GTPase effector N-WASP in neurite outgrowth. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Rho family GTPase proteins Cdc42, Rac and Rho play critical roles in signalling pathways leading to cytoskeletal rearrangements and involved in cell growth and differentiation. In neuronal cells, the combined action of Cdc42 and Rac results in the formation of neurites, whereas Rho acts to collapse neurites and growth cones. Overexpression of the Cdc42/Rac effector proteins N-WASP, IRS-58 and Pak (the latter with CAAX motif to promote membrane localisation) all promoted neurite outgrowth in neuroblastoma N1E-115 cells, with differing morphologies. Combinations of these effector molecules did not act synergistically in the promotion of neurite outgrowth. Overexpression of the downstream effectors of Pak, PIX and GITl, inhibited outgrowth by the effectors N-WASP and IRS-58, instead they promoted cell motility. N-WASP is comprised of several domains and the C-terminal (VCA) region has been shown to interact with the Arp2/3 complex, F-actin and to be involved in the formation of the autoinhibited structure. The verprolin homology domain (VH), which is believed to be responsible for the binding/severing of actin, inhibits neurite outgrowth induced by the effectors IRS-58, N-WASP, PakCAAX and low serum conditions, but does not inhibit filopodia formation. Live imaging studies of N1E-115 cells overexpressing N-WASP suggest its importance in filopodia turnover. Microinjection of NWASP-/- cells with IRS-58 observed in real time indicates the importance of N-WASP the formation of filopodia by IRS-58. These data together suggest the existence of a balance between the induction of cell motility or neurite outgrowth by different Rho GTPase effectors. The observations made in this project indicate the requirement of N-WASP in neurite outgrowth and of IRS-58's formation of filopodia.

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