82WiskottAldrich syndrome protein (WASP), the gene mutated in the haematological disorder Wiskott-Aldrich syndrome, is the founding member of a family of conserved cytoskeletal regulators. WASP family proteins regulate actin dynamics through binding and activation of the Arp2/3 complex, which nucleates new actin filaments. Recently, a huge amount of information on WASPs has become available, both in terms of the function of individual domains, identification of their binding partners, and the unravelling of complex regulatory mechanisms. Together, these new findings place WASP-related molecules at the crossroads between distinct signalling pathways, which they integrate into coherent cytoskeletal responses. Addresses MRC-LMCB and Cell Biology Unit, University College London, Gower Street, London WC1E 6BT, UK; e-mail: e.caronucl.ac.ukCurrent Opinion in Cell Biology 2002, 14:82870955-0674/02/$ see front matter 2002 Elsevier Science Ltd. All rights reserved.Abbreviations BRbasic region GBDGTPase-binding domain N-WASPneuronal WASP PIP2phosphatidylinositol 4,5-bisphosphate SCARsuppressor of cAMP receptor SHSCAR-homology WASWiskottAldrich syndrome WASPWiskottAldrich syndrome protein WH1WASP-homology domain 1 WH2/C/AWASP-homology 2/Central/Acidic regionsIntroduction Actin cytoskeleton dynamics drive most aspects of eukary- otic cell biology, including growth factor-induced cell shape changes, cell division, some endocytic processes (e.g. phagocytosis) and motile behaviours such as locomotion and chemotaxis. The haematopoietic cells of patients with the WiskottAldrich syndrome (WAS) are characterised by morphological abnormalities and impaired actin cytoskeleton remodelling in response to stimulation 1. The Wiskott- Aldrich syndrome protein (WASP), encoded by the WAS gene, is the prototype of a family of homologous, conserved molecules that link signalling pathways to the actin cyto- skeleton by virtue of their ability to activate Arp2/3-mediated de novo actin polymerisation 2. Recent findings have increased our understanding of the complex intra- and inter- molecular interactions that regulate the function of WASP- family molecules both in vitro and in biological processes. Getting intimate with WASP: new structural and biochemical insights The WASP family members (five in total in mammals: WASP, neuronal WASP N-WASP, and three suppressor ofcAMP receptor SCAR isoforms) share two main regions of homology: a central segment rich in proline residues and a carboxy-terminal module made of three characteristic domains WASP-homology 2/Central/Acidic regions (WH2/C/A). These features are also found in the unique homologues, Bee1 (Las17) and Wsp1, of Saccharomyces cerevisiae and S. pombe, respectively (Figure 1). The amino-terminal half of the molecule defines two functional subgroups, based on the presence of either a WASP-homology (WH) domain 1 (found in WASP, N-WASP, and the yeast WASP-related molecules) or a SCAR-homology (SH) domain, specific to SCAR isoforms. The past two years have witnessed an explosion in the number of studies that address the role of these various domains and report the identification of binding partners.Mechanisms of Arp2/3 activation The carboxyl terminus of WASP family members initiates the growth of new actin filaments by bringing together actin monomers and Arp2/3 in solution or to the side of pre-existing actin filaments. It was already known that an isolated WH2/C/A module binds actin monomers and Arp2/3, and can constitutively activate nucleation and branching in a cell-free assay 3,4. The relative contributions of the WH2, C and A domains have now been been studied in detail. In WASP, both the WH2 and C domains contribute to high-affinity binding of actin monomers 5. Whether the same is true for N-WASP, the sole WASP family member with two WH2 domains is unknown at present; however, it has been suggested that the presence in N-WASP of two adjacent WH2 domains might be sufficient to enable strong actin binding, without a major contribution for the C domain in G-actin binding 6. Likewise, both the C and A domains contribute to optimal Arp2/3 binding in vitro. Residues that are crucial for actin and Arp2/3 binding were mapped. Interestingly, one point mutant (R477K) showed normal binding properties but a reduced ability to activate Arp2/3-mediated actin nucleation, suggesting the existence of an activation step. The structural basis for this potential regulatory mechanism is currently unknown. Finally, it has now been demonstrated that actin filaments enhance the binding of the Arp2/3 complex to the WH2/C/A module, suggesting that nucleation by filament-bound Arp2/3 is favoured over that by free Arp2/3 (5; see also Update). Autoinhibitory interactions Ectopic expression of full-length WASP and N-WASP induces the formation of actin clusters in cells but does not lead to major changes in cell morphology 79, suggesting that WASP and N-WASP are intrinsically in