MICROSCOPY RESEARCH AND TECHNIQUE 47:107–113 (1999) Wiskott-Aldrich Syndrome: A Disorder of Haematopoietic Cytoskeletal Regulation ADRIAN J. THRASHER* AND SIOBHAN BURNS Molecular Immunology Unit, Institute of Child Health, London WC1N 1EH, United Kingdom KEY WORDS WASp; SCAR; Arp2/3; chemotaxis; filopodia; macrophage; dendritic cell ABSTRACT The Wiskott-Aldrich Syndrome (WAS) is a rare inherited X-linked recessive disease characterised by immune dysregulation and microthrombocytopenia. Recently, the biological mechanisms that are responsible for the pathophysiology of WAS have been shown to be linked to the regulation of the actin cytoskeleton in haematopoietic cells. The WAS protein (WASp) is now known to be a member of a unique family that share similar domain structures, and that are responsible for transduction of signals from the cell membrane to the actin cytoskeleton. The interactions between WASp, the Rho family GTPase Cdc42, and the cytoskeletal organising complex Arp2/3 are probably critical to many of these functions, which, when disturbed, translate into measurable defects of cell polarisation and motility. Microsc. Res. Tech. 47:107–113, 1999. r 1999 Wiley-Liss, Inc. PHENOTYPIC FEATURES OF THE WISKOTT-ALDRICH SYNDROME The Wiskott-Aldrich Syndrome (WAS) is a rare inherited X-linked recessive disease characterised by immune dysregulation and microthrombocytopenia (Wiskott, A, 1939; Aldrich, 1954; Remold O’Donnell, 1996; MIM 301000). The clinical phenotype of the immune disorder includes susceptibility to pyogenic, viral, and opportunistic infections, and eczema (Sullivan, 1994). Unlike thrombocytopenia, which is present at birth, the immunological manifestations are progressive, with decreasing numbers of T lymphocytes (particularly CD8 cells) during early childhood, defects in proliferative and delayed type hypersensitivity responses, and deficient production of antibodies to both polysaccharide and protein antigens. Later complications to arise from this process include autoimmune and lymphoproliferative disease. In its mildest form, known as X-linked thrombocytopenia (XLT), mutations in the same gene produce the characteristic platelet abnormality but minimal immunological disturbance (Villa, 1995; MIM 313900). WISKOTT-ALDRICH SYNDROME PROTEIN (WASp) IS A MEMBER OF A LARGER FAMILY Although abnormalities of platelet structure, and the presence of dysmorphic microvilli on the surface of WAS T lymphocytes and T lymphocyte cell lines, suggested an underlying cytoskeletal abnormality, it was not until the gene was cloned in 1994 (gene bank accession number U12707) that it became possible to examine intimate biochemical interactions within the cell (Derry et al., 1994; Gallego et al., 1997; Kenney et al., 1986; Molina et al., 1992). The WAS gene is now known to encode a 502 amino acid proline-rich intracellular protein (WASp) expressed exclusively in haematopoietic cells, which belongs to a recently defined family of more widely expressed proteins involved in transduction of signals from receptors on the cell surface to the actin cytoskeleton. Other members of the family inr 1999 WILEY-LISS, INC. clude neural (N)-WASp, SCAR (suppressor of G-protein coupled cyclic-AMP receptor cAR, isolated from Dictyostelium), four human SCAR proteins (hsSCAR1–4), other homologues of SCAR (in mouse, Caenorhabditis elegans, and Drosophila), and the WASp-related S. cerevisiae protein Las17p/Bee1p (Bear et al., 1998; Li, 1997; Miki et al., 1996). DOMAIN ORGANISATION OF WASp AND RELATED PROTEINS WASp, N-WASp, SCAR, and Las17p/Bee1p are organised into modular domains defined by sequence homology and binding interactions (see Fig. 1). They all posses C-terminal polyproline ‘‘P’’, Wiskott-homology (WH)-2 and acidic ‘‘A’’ domains. N-WASp, has a similar domain structure to WASp, but has an additional IQ motif in the N-terminal half of the protein, which binds calmodulin light chains. Although first identified as a Grb2-binding protein in bovine brain, N-WASp is widely expressed (Miki et al., 1996). SCAR family proteins are homologous to each other in the N-terminus (SCAR homology domain, SHD), but differ in this region from WASp and N-WASp and lack typical PH and CRIB domains. However, they retain a basic domain ‘‘B’’ upstream of the ‘‘P’’ region (Bear et al., 1998). Las17p/ Bee1p has a similar domain structure to WASp and N-WASp except for the absence of a recognisable CRIB domain (Li, 1997). Pleckstrin Homology (PH) Domain For N-WASp, a Pleckstrin homology (PH)-domain has been assigned to the N-terminus overlapping a WASp-homology1 (WH1) 1-domain. The WH1 domain has been identified in several proteins including vasodilator-stimulated phosphoprotein (VASP), Dena (Drosophila-enabled), Mena, Homer, and Ev1, all of which *Correspondence to: Adrian J. Thrasher, Molecular Immunology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. E-mail: email@example.com Received 11 Aprril 1999; accepted in revised form 25 May 1999 108 A.J. THRASHER AND S. BURNS Fig. 1. Domain structure of WASp: see text for full explanation. For the related protein, N-WASp, a putative N-terminal Pleckstrin homology (PH)-domain, binds to the acidic phospholipid phosphatidylinositol (4,5)-bis-phosphate (PIP2), and could therefore facilitate localisation to the cell membrane. An undefined region in the N-terminal half also binds to Wiskott-interacting protein (WIP), which itself may be regulated by Src-homology (SH)-3 containing signaling molecules, and may also bind to actin directly. One of the most important functions of WASp, mediated through a Cdc42/Rac interactive binding (CRIB) motif, is to act as an effector for the cytoskeletal regulator Cdc42, which is known to induce the formation of focal adhesion complexes have been implicated in the regulation of the actin cytoskeleton (Callebaut et al., 1998; Gertler et al., 1996; Ponting and Phillips, 1997). Although the function of the PH-domain has not been defined for WASp, this type of domain may well be important for interaction with other proteins or lipids. For the related protein N-WASp, the N-terminal PH-domain has been shown to bind the acidic phospholipid phosphatidylinositol (4,5)bis-phosphate (PIP2), and through this interaction could allow N-WASp or WASp to localise at the cell membrane (Lemmon et al., 1996; Miki et al., 1996). However, careful alignment of the WASp PH-domain to a defined consensus has recently shown that only 9 out of 94 amino acids are identical, casting doubt on its existence (Insall and Machesky, in press; Musacchio et al., 1993). Furthermore, the related protein Las17p/ Bee1 cannot contain a PH-domain as the initiation codon occurs at a site within the PH-domain itself (Insall and Machesky, in press). and filopodial extensions. The activity of WASp may also be critically regulated by cytosolic signaling molecules binding to a proline-rich region (PPPPP). The effector function of WASp is probably mediated through the C-terminal region, which includes a Wiskott homology domain (WH2), and an acidic ‘‘A’’ region that binds to p21-Arc of the Arp2/3 complex. In the inactive state, the ‘‘A’’ region may form an intramolecular interaction with a basic ‘‘B’’ region. In the event of activation, the ‘‘A’’ region would become available for interaction with the Arp2/3 complex, which, it is suggested, mediates the assembly of branching filaments from monomeric actin bound to the WH2 domain. CDC42/Rac Interactive Binding (CRIB) Motif The first real biochemical evidence to implicate WASp in the regulation of the actin cytoskeleton came from studies in which WASp was shown to cluster physically with polymerised actin. In addition, WASp bound specifically to the GTP-bound form of the Rho-like GTPase Cdc42, and less well to GTP-bound Rac, suggesting that it acts as a direct effector molecule for Cdc42 (Aspenstrom et al., 1996; Kolluri et al., 1996; Symons et al., 1996). Cdc42 belongs to the Rho family of small GTPbinding proteins, and regulates the formation of distinct actin-filament containing protrusions known as filopodia in fibroblast and monocytic cell lines (Allen et al., 1997; Kozma et al., 1995; Nobes and Hall, 1995). More recently, N-WASp has been shown to potentiate the formation of Cdc42-induced filopodia in microinjected COS and Swiss 3T3 cells (Miki et al., 1998). In contrast, growth factor-induced activation of the re- ROLE OF WASp IN CYTOSKELETAL FUNCTIONS. lated GTP-binding protein Rac leads to accumulation of an actin network at the cell periphery producing lamellapodia and membrane ruffling (Lamarche et al., 1996; Ridley et al., 1992). Cdc42 and Rac have also been shown to participate in the formation of cell-substratum focal adhesion complexes distinct from Rhoinduced focal adhesions (Nobes and Hall, 1995). For both WASp and N-WASp, the interaction with Cdc42/ Rac has been shown to be mediated through a Cdc42/ Rac small GTPase interactive binding (CRIB) motif (or GTPase binding domain, GBD), which is found in many downstream effectors of Cdc42 and Rac, although regions outside this motif are necessary for structure formation and for tight interaction (Rudolph et al., 1998). Some clues to the cellular pathophysiology of WAS can, therefore, be obtained from work, implicating Cdc42 both as a regulator of actin-containing filopodial extensions, as well as in the development of cell polarity in budding yeast and murine T lymphocyte hybridoma cells, Fc-receptor mediated phagocytosis, and macrophage chemotaxis (Adams et al., 1990; Allen et al., 1998; Caron and Hall, 1998; Johnson and Pringle, 1990; Kozma et al., 1995; Massol et al., 1998; Nobes and Hall, 1995; Stowers et al., 1995). Proline-Rich ‘‘P’’ Domain WASp has been shown by several groups to bind SH3 domains of cytosolic signalling molecules, although the significance of many of these in vitro associations is unclear (Brickell et al., 1998). More convincingly, in vivo binding interactions have been shown for the adapter proteins Nck and Grb2, the protein tyrosine kinase Fyn, a cytoplasmic protein-tyrosine kinase of the c-Src family, which may participate in regulation of cytoskeletal architecture, and the B cell Tec family tyrosine kinase, Btk (Baba, 1999; Banin et al., 1996; Rivero et al., 1995; She et al., 1997). More recently, a cytoskeletal-associated protein related to Schizosaccharomyces pombe cytokinetic cleavage furrow regulatory protein (CDC15p), known as proline-serine-threonine phosphatase-interacting protein (PSTPIP), has been shown to interact directly with WASp and to inhibit WASp-induced actin bundling activity in vivo in a process that is regulated by tyrosine phosphorylation of the SH3 domain (Wu et al., 1998). The interaction between WASp and SH3 domains is mediated by polyproline P stretches rich in the SH3 binding motif PXXP. WASp Interacting Protein (WIP) Another proline-rich protein, WASp-interacting protein (WIP), has recently been shown to co-immunoprecipitate with WASp from lymphocyte cell lysates (although it is ubiquitously expressed, and may, therefore, also interact with other members of the WASp family) (Ramesh et al., 1997). Two verprolin homology domains at the N-terminus of WIP, suggest that it may interact directly with actin, while the presence of SH3-binding motifs could provide a link with other cellular signalling pathways. The region of binding to WASp has not been accurately identified, although it appears to require N-terminal sequences distinct from the CRIB domain. 109 Carboxy Terminal Binding Domains The carboxy terminal regions of WASp, N-WASP, and SCAR contain an actin-binding region known as WH2 or verprolin homology (VH) domain, and a cofilin homology domain that may participate in depolymerisation of actin filaments (Miki et al., 1996, 1998). Carboxyterminal of the WH2 domain lies a conserved series of acidic residues ‘‘A’’, which recently have been shown to be necessary for the binding (probably mediated by p21-Arc) of actin-related proteins of a cytoskeletal organiser known as the Arp2/3 complex (Machesky and Insall, 1998). The Arp2/3 complex consists of 7 proteins including the actin-related proteins Arp2 and Arp3, and other components p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (Welch et al., 1997a). This complex is localised to areas of active actin polymerisation, such as in lamellipodia and at the base of filopodia. It has also been shown to initiate actin polymerisation on the surface of the motile intracellular pathogen Listeria Monocytogenes and may, therefore, be essential for the initiation or formation of actin tails that propel the organism through the cytosol (Welch et al., 1997b). In vitro the complex has been shown to nucleate new actin filaments, a process that is considerably enhanced when bound to WASp-family proteins (Machesky and Insall, 1998; Machesky et al., 1999; Mullins et al., 1998; Rohatagi et al., 1999). Overexpression of the conserved C-terminal acidic domain (but not the WH2 domain alone) of WASp and SCAR disrupts the localisation of the Arp2/3 complex, inhibits lammellipodia and stress fibre formation in Swiss 3T3 cells in response to plateletderived growth factor (PDGF) and sphingosine 1-phosphate, respectively, and inhibits endogenous filopodia formation in J774 macrophages, suggesting that WASprelated proteins regulate the cytoskeleton through this complex. It is, therefore, possible that the Arp2/3 complex becomes activated on binding to WASp and mediates the regulated assembly of actin filaments from monomeric actin bound to the WH2 domain of WASp. An additional possibility is that the C-terminal acidic domain binds to a basic domain upstream of CRIB (in WASp and N-WASp) and upstream of the ‘‘P’’ region (in SCAR), thereby masking binding sites for Arp2/3, which only become available when GTP-bound Cdc42 or relevant SH3 domains interact with WASp (Miki et al., 1998). WASp family proteins also interact with the small actin monomer binding protein profilin, although the nature of this interaction in terms of the binding residues, and whether it is direct or mediated through an intermediate such as the Arp2/3 complex are unknown. Cytoskeletal Disturbances in WAS The generation of regulated immune responses is dependent on the ability of cells to migrate in response to integrated chemical signals (reviewed in Baggiolini, 1998). In addition to the influx of effector cells to sites of inflammation, these processes are responsible for the relocation of dendritic cells from their surveillance positions in non-lymphoid tissue to the secondary lymphoid organs, and migration of lymphocytes into specialised B and T cell zones (Banchereau and Steinmann, 1998). The maintenance of a normal immune repertoire 110 A.J. THRASHER AND S. BURNS also depends on the ability of thymic emigrants to interact in some way with MHC molecules expressed on peripheral dendritic cells, and the same may be true for the persistence of mature B cells, although the existence and location of a ligand to provide the necessary B cell receptor signal remains uncertain (Brocker et al., 1997; Lam et al., 1997; Rooke et al., 1997; Takeda et al., 1996; Tanchot et al., 1997). The cell biological mechanisms that allow these trafficking processes to occur are, therefore, intimately related to the regulation of the cytoskeletal architecture, and the response of this to external stimuli is of fundamental importance to immune cell homeostasis, and the initiation of inflammatory processes. Cells from patients with WAS provide natural models with which some questions about cytoskeletal regulation can be answered. While mutations restricted to the first three exons of the WAS gene may lead to a mild clinical phenotype and expression of residual protein, in the majority of cases WASp is destabilised, and most patients are effectively null mutants (MacCarthy-Morrogh et al., 1998). Abnormalities of Immune Cell Chemotaxis and Chemokinesis Disturbance of cellular processes that are directly related to re-organisation of cytoskeletal architecture, such as those directing cell motility, could contribute significantly to the immunopathology of WAS. To investigate the effects of WAS mutation on cell motility, we used a direct-viewing chemotaxis chamber to analyse chemotactic responses of WAS neutrophils and macrophages migrating on serum-coated glass surfaces in stable linear concentration gradients of diffusable attractants. Chemotaxis of macrophages (to CSF-1) but not of neutrophils (to fMLP or IL-8) was found to be abolished in all cases, whereas the speed of random motility of both cell types was found to be indistinguishable from control cells (Zicha et al., 1998). Similar disturbances of motility in WAS cells have been reported elsewhere (Badolato et al., 1998). Furthermore, polymerisation of actin on the ventral surface of WAS macrophages and extension of filopodia were both found to be severely compromised, adding further support to the role of WASp as an effector for Cdc42 in haematopoietic cells (Thrasher and Jones, unpublished results). Interestingly, Bac1 rodent macrophages microinjected with a dominant negative Cdc42 mutant (N17Cdc42) have been shown to have specific defects of polarisation and chemotaxis identical to those of WAS mutant macrophages (Allen et al., 1998). The effects of this disturbance on the localisation of Arp2/3 complex are currently being investigated. The apparent functional normality of neutrophils in terms of both chemotaxis and chemokinesis could be explained by the use of alternative WASp family proteins such as SCAR for G-protein coupled signalling, and is supported by the fact that expression of WASp is virtually undetectable in this cell type (Thrasher, unpublished results). A major contribution to the immune dysregulation of WAS could arise from the inability of specialised antigen presenting cells known as dendritic cells (DCs) to traffic normally between non-lymphoid tissues and T lymphocyte-rich areas of lymphoid organs (Banchereau and Steinmann, 1998; Thrasher et al., 1998). In similar motility experiments to those conducted on macro- phages and neutrophils, WAS DCs exhibited major abnormalities in the distribution of the peripheral filamentous F-actin cytoskeleton (see Fig. 2), failed to develop normal polarised morphologies or to extend dendritic processes on a fibronectin substratum, and were essentially immotile when stimulated with fMLP or RANTES (Binks et al., 1998). In contrast, formation of membrane ruffles, presumably mediated by Rac, appeared to be quantitatively and qualitatively preserved. WAS macrophages tested under the same conditions showed identical defects in cell motility, suggesting that both these cell types are compromised at least at two levels (Thrasher and Jones, unpublished data). Firstly, the cells are unable to respond to chemical stimuli in a directional manner, even though they appear to activate normally. These disturbances probably relate to abnormalities of Cdc42-WASp-mediated filopodia formation, and are consistent with recent suggestions that these structures are essential for macrophage chemotaxis (Allen et al., 1997, 1998). At a second level, WAS macrophages and DCs polarise poorly and are unable to translocate when adherent to surfaces that require engagement of integrins on the cell surface. The precise mechanisms by which WASp directs this process are unclear at present, but dysfunctional interaction between anchoring integrins, the cytoskeleton, and intracellular myosin-generated forces, may restrict the capacity of the cell to achieve both spatial asymmetry and contractile movement. As WASp is expressed in all haematopoietic cells, similar abnormalities of cell traffic may be apparent in other haematopoietic lineages. For lymphocytes, the gross morphological disturbances of surface microvilli suggest that dysmotility similar to that of macrophages and DCs could also be present. Preliminary data obtained from WASp mutant mice generated by gene targeting suggests that this may well be the case (Snapper et al., 1998; Snapper and Rosen, 1999). In addition, WAS carrier females almost universally exhibit non-random X-inactivation patterns in CD34⫹ progenitors, indicating that WASp must also be functional even at this level. On the basis of our own experiments that demonstrate that WASp is expressed in intra-aortic CD34⫹ cell clusters at the aorta-gonadmesonephros (AGM) stage of human embryonic haematopoiesis, WAS stem cells may be less able than their normal equivalents to seek out the appropriate microenvironmental niches in which liver, and later bone marrow, haematopoiesis is established (Thrasher and Marshall, unpublished results). This is reminiscent of mice lacking the chemokine receptor CXCR4 or its ligand SDF-1, which have markedly impaired colonisation of bone marrow by multipotent haematopoietic cells, and murine ␤1 integrin –/– integrin chimeras, which do not develop definitive liver haematopoiesis due to a failure to seed this tissue during embryonic development (Fassler and Meyer, 1995; Nagasawa et al., 1996; Zou et al., 1998). Abnormalities of Other Cytoskeletal Related Functions In addition to gross defects of cell motility, it is likely that WASp is important for the regulation of the cytoskeleton in other processes such as lymphocyte activation and phagocytosis. For example, Cdc42- ROLE OF WASp IN CYTOSKELETAL FUNCTIONS. 111 Fig. 2. WAS dendritic cells. a: WAS DCs derived in vitro from CD14⫹ peripheral blood precursors and stained with TRITC-phalloidin on a fibronectin substratum. Compared to normal DCs (b), WAS cells have a poorly defined cytoskeletal structure, and lack typical podosomal structures on the ventral surface. mediated actin polymerisation has also been shown to be necessary for promoting the correct orientation of lymphocytes and antigen presenting cells (Stowers et al., 1995). Following activation of lymphocytes, an asymmetric assembly of receptors and signalling molecules known as the Cap forms on the cell surface (reviewed in Penninger and Crabtree, 1999). In addition, interaction between T lymphocytes and antigen- presenting cells results in the formation of highly organised clusters of antigen receptors, coreceptors, adhesion, and signalling molecules (supramolecular activation clusters, SMAC), a process that requires actin polymerisation. The segregation of these molecules within SMACs is critical for the initiation of physiological immune responses. T cells from WAS patients and WAS mutant mice generated by gene 112 A.J. THRASHER AND S. BURNS targeting both show abnormalities of antigen receptor stimulated proliferation, and in the mice of antigen receptor Cap formation (Molina et al., 1993; Snapper et al., 1998). In contrast, studies on murine WAS B cells have demonstrated normal proliferation, and normal Cap formation, suggesting that there may be functional redundancy in this cell type, although defects in proliferative responses of human WAS B cells have been reported previously (Facchetti et al., 1998; Simon et al., 1992; Snapper et al., 1998). Recently, several groups have shown that Fc␥ receptor (Fc␥R)-mediated phagocytosis is regulated by Cdc42 and Rac1 (Caron and Hall, 1998; Cox et al., 1997; Massol et al., 1998;). It also seems likely that Cdc42 and Rac1 have distinct non-overlapping functions that act co-operatively in the formation of the phagocytic cup. In one study, two distinct mechanisms of phagocytosis were identified (Caron and Hall, 1998). One used by the immunoglobulin receptor Fc␥R was mediated by Cdc42 and Rac1 whereas that used by the complement receptor (CR3) was mediated by Rho. In addition, microinjection of the WASp CRIB domain (as a competitor for Cdc42 effectors) into J774 macrophages completely prevented Fc␥R-mediated phagocytosis. More recently, we have found that Fc␥R-mediated phagocytosis is impaired in WAS macrophages, but remains intact in neutrophils, adding further support to the suggestion that related proteins are functional in this cell type (Thrasher and Lorenzi, unpublished data). Platelet Abnormalities in WAS The mechanism of the platelet defects in WAS are largely unknown. However, it appears likely that WASp is involved in the formation of platelets from megakaryocytes, in the maintenance of platelet integrity in the circulation, and in platelet aggregation at sites of tissue injury. As shown for other cell types in WAS, these processes may be linked to abnormalities of filopodial extension. CONCLUSIONS The ability to study WAS mutant cells provides a unique opportunity to unravel some of the mechanisms of actin polymerisation in the haematopoietic system. The cellular phenotype of WAS clearly demonstrates that defects in the regulation of cytoskeletal architecture have profound effects on the motile and morphological characteristics of cells that initiate and regulate immune responses. The extent to which these defects operate in vivo is under investigation, and will be greatly facilitated by the development of murine models of WAS. In addition, processes unrelated to chemokinesis, for example, antigen-mediated lymphocyte activation and phagocytosis of either microorganisms or apoptotic cells, may be critically dependent on normal WASp function. Understanding the way in which WASp participates in these processes will enhance our understanding of cytoskeletal biology, and hopefully will translate into the development of new treatments for this disease. 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