Research Article 3535 Introduction Neurite outgrowth is essential for the proper formation of neuronal networks in the developing nervous system and is ultimately dependent on cytoskeletal dynamics, which often requires the activity of the low molecular weight GTPases of the Rho family. Rho GTPases are molecular switches, which, upon binding GTP, undergo conformational changes that enable the regulation of downstream target proteins (Jaffe and Hall, 2005). Hydrolysis of GTP to GDP turns off and limits the extent of downstream signalling. Members of the Rho family of GTPases, which include RhoA, Rac1 and Cdc42 have been extensively studied in the context of neurite remodelling (Govek et al., 2005). Neurite dynamics, controlled by Rho GTPase, have been widely studied in model systems such as the neuronal cell line N1E-115 (Gebbink et al., 1997; Hirose et al., 1998; Jalink et al., 1993; Jalink et al., 1994; Kozma et al., 1997; Kranenburg et al., 1999; Postma et al., 1996). These cells maintain a spherical morphology in the presence of serum; however, withdrawal from serum results in rapid cell flattening, differentiation and the formation of neurites. Stimulation of serum-starved cells with different repulsive agonists, such as lysophosphatidic acid (LPA), trigger a rapid collapse of neurites that is dependent upon the activation of RhoA. Using N1E-115 cells, we and others reported that activity of phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) is essential for neurite retraction in response to LPA, thrombin and sphingosine 1-phosphate (van Horck et al., 2002; Yamazaki et al., 2002). In isolated hippocampal neurones, overexpression of PIP5K also suppresses axonal growth induced by ARF6 inhibition (Hernandez- Deviez et al., 2004). PIP5Ks are regulated by Rac (Hartwig et al., 1995; Tolias et al., 1998; Tolias et al., 2000; van Hennik et al., 2003), which prompted an exploration of a role for Rac in the regulation of PIP5Ks and PtdIns(4,5)P 2 in neuronal retraction. By defining the molecular interaction sites between the Rac and PIP5K, we have generated a PIP5K mutant that no longer interacts with endogenous Rac. We have used this and other mutants of Rac to demonstrate that the interaction between Rac and PIP5K is essential for the proper localisation of PIP5K and synthesis of phosphatidyl- inositol(4,5)bisphosphate [PtdIns(4,5)P 2 ]. Finally, we found that the interaction between PtdIns(4,5)P 2 and vinculin, a focal- adhesion-stabilising protein, is required for PIP5K and LPA- mediated neurite retraction. Rac controls PIP5K localisation and PtdIns(4,5)P 2 synthesis, which modulates vinculin localisation and neurite dynamics Jonathan R. Halstead 1, *, Nicolai E. Savaskan 1,2 *, Iman van den Bout 3 , Francis Van Horck 4 , Amra Hajdo-Milasinovic 1 , Mireille Snell 1 , Willem-Jan Keune 1,3,‡ , Jean-Paul ten Klooster 5 , Peter L. Hordijk 5 and Nullin Divecha 1,3,‡,§ 1 Division of Cell Biology, The Netherlands Cancer Institute Amsterdam, Amsterdam, 1066 CX, The Netherlands 2 Institute of Cell Biology and Neurobiology, Charité – Universitätsmedizin Berlin, D-10115 Berlin, Germany 3 The CRUK Inositide Laboratory, The Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, UK 4 Department of Physiology, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK 5 Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, 1066 CX, The Netherlands *These authors contributed equally to this work ‡ Present address: The CRUK Inositide Laboratory, The Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, UK § Author for correspondence (ndivecha@picr.man.ac.uk) Accepted 16 June 2010 Journal of Cell Science 123, 3535-3546 © 2010. Published by The Company of Biologists Ltd doi:10.1242/jcs.062679 Summary In N1E-115 cells, neurite retraction induced by neurite remodelling factors such as lysophosphatidic acid, sphingosine 1-phosphate and semaphorin 3A require the activity of phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks). PIP5Ks synthesise the phosphoinositide lipid second messenger phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P 2 ], and overexpression of active PIP5K is sufficient to induce neurite retraction in both N1E-115 cells and cerebellar granule neurones. However, how PIP5Ks are regulated or how they induce neurite retraction is not well defined. Here, we show that neurite retraction induced by PIP5K is dependent on its interaction with the low molecular weight G protein Rac. We identified the interaction site between PIP5K and Rac1 and generated a point mutant of PIP5K that no longer interacts with endogenous Rac. Using this mutant, we show that Rac controls the plasma membrane localisation of PIP5K and thereby the localised synthesis of PtdIns(4,5)P 2 required to induce neurite retraction. Mutation of this residue in other PIP5K isoforms also attenuates their ability to induce neurite retraction and to localise at the membrane. To clarify how increased levels of PtdIns(4,5)P 2 induce neurite retraction, we show that mutants of vinculin that are unable to interact with PtdIns(4,5)P 2 , attenuate PIP5K- and LPA-induced neurite retraction. Our findings support a role for PtdIns(4,5)P 2 synthesis in the regulation of vinculin localisation at focal complexes and ultimately in the regulation of neurite dynamics. Key words: Lipid kinase, PIP5K, Rac, G-proteins, Neurite retraction, PtdIns(4,5)P 2 , PIP2 Journal of Cell Science