The International Journal of Biochemistry & Cell Biology 34 (2002) 806–815 Restoration of podosomes and chemotaxis in Wiskott–Aldrich syndrome macrophages following induced expression of WASp Gareth E. Jones a,∗ , Daniel Zicha b , Graham A. Dunn c , Mike Blundell d , Adrian Thrasher d a The Randall Centre, King’s College London, London SE1 1UL, UK b Imperial Cancer Research Fund, London WC2A 3PX, UK c MRC Muscle and Cell Motility Unit, King’s College London, London SE1 1UL, UK d Institute of Child Health, University College London, London WC1N 1EH, UK Received 10 September 2001; received in revised form 21 November 2001; accepted 26 November 2001 Abstract We used a direct-viewing (Dunn) chemotaxis chamber to analyse the chemotactic responses of human normal and Wiskott–Aldrich syndrome (WAS) macrophages to the cytokine colony stimulating factor-1 (CSF-1). In five patients with classic WAS, where specialised adhesion complexes called podosomes are absent, chemotaxis of macrophages was abolished. The deficient chemotactic responses of WAS macrophages following cytokine stimulation could be correlated with abnor- malities in cell polarisation and actin organisation. In a series of cell microinjection studies we found that normal chemotactic responses were restored in WASp macrophages transfected with a full-length human WAS construct. Expression of exoge- nous WAS protein (WASp) in these cells also restored normal polarised cell morphology and the ability to form podosomes. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Actin cytoskeleton; Podosome; Cell migration; Cytokine; Chemotaxis; WASp 1. Introduction The Wiskott–Aldrich syndrome (WAS) gene en- codes a 502 amino acid proline-rich intracellular WAS protein (WASp) expressed exclusively in haematopoi- etic cells [1]. It belongs to a recently-defined family of more widely expressed proteins involved in the transduction of signals from receptors on the cell surface to the actin cytoskeleton [2]. Other members of the WASp family include neural (N)-WASp, sup- pressor of G-protein coupled cyclic-AMP receptor ∗ Corresponding author. Tel.: +44-20-7848-6466; fax: +44-20-7848-6435. E-mail address: gareth.jones@kcl.ac.uk (G.E. Jones). (cAR) SCAR, isolated from Dictyostelium, three human SCAR proteins (hsSCAR1-3), other homo- logues of SCAR (in mouse, Caenorhabditis elegans, and Drosophila), and the WASp-related S. cerevisiae protein Las17p/Bee1p. WASp, N-WASp, SCAR, and Las17p/Bee1p are organised into modular domains de- fined by sequence homology and binding interactions [3,4]. Deficiency of WASp results in WAS, a rare in- herited X-linked recessive disease characterised by im- mune dysregulation and microthrombocytopenia [5]. Biochemical evidence to implicate WASp in the regulation of the actin cytoskeleton was first provided by studies suggesting that WASp acts as a direct effector molecule for Cdc42 [6–8]. Cdc42 belongs to the Rho family of small GTP-binding proteins, 1357-2725/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII:S1357-2725(01)00162-5