Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP

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Abstract

Cdc42 is a small GTPase of the Rho family which regulates the formation of actin filaments to generate filopodia1,2. Although there are several proteins such as PAK3, ACK4 and WASP (Wiskott–Aldrich syndrome protein)5 that bind Cdc42 directly, none of these can account for the filopodium formation induced by Cdc42. Here we demonstrate that before it can induce filopodium formation, Cdc42 must bind a WASP-related protein, N-WASP, that is richest in neural tissues6 but is expressed ubiquitously. N-WASP induces extremely long actin microspikes only when co-expressed with active Cdc42, whereas WASP, which is expressed in haematopoietic cells, does not, despite the structural similarities between WASP and N-WASP. In a cell-free system, addition of active Cdc42 significantly stimulates the actin-depolymerizing activity of N-WASP, creating free barbed ends from which actin polymerization can then take place. This activation seems to be caused by exposure of N-WASP's actin-depolymerizing region induced by Cdc42 binding.

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Figure 1: Enhancement of Cdc42-induced filopodium formation by N-WASP but not by WASP.
Figure 2: Specific and direct binding of N-WASP to activated Cdc42 through the GBD/CRIB motif.
Figure 3: Requirement for endogenous N-WASP for microspike formation in Swiss 3T3 cells.
Figure 4: Effect of Cdc42 on depolymerization of actin filaments by N-WASP in a cell free system.

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Acknowledgements

We thank S. Suetsugu and Y. Banzai for their skilful technical assistance. This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture of Japan and a Grant-in-Aid for Research for the Future Program from the Japan Society for the Promotion of Science.

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Correspondence to Tadaomi Takenawa.

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