The Wiskott-Aldrich syndrome protein (WASp) is a key cytoskeletal regulator in hematopoietic cells. Covalent modification of a conserved tyrosine by phosphorylation has emerged as an important potential determinant of activity, although the physiological significance remains uncertain. In a murine knockin model, mutation resulting in inability to phosphorylate Y293 (Y293F) mimicked many features of complete WASp-deficiency. Although a phosphomimicking mutant Y293E conferred enhanced actin-polymerization, the cellular phenotype was similar due to functional dysregulation. Furthermore, steady-state levels of Y293E-WASp were markedly reduced compared to wild-type WASp and Y293F-WASp, although partially recoverable by treatment of cells with proteasome inhibitors. Consequently, tyrosine phosphorylation plays a critical role in normal activation of WASp in vivo, and is indispensible for multiple tasks including proliferation, phagocytosis, chemotaxis, and assembly of adhesion structures. Furthermore, it may target WASp for proteasome-mediated degradation, thereby providing a default mechanism for self-limiting stimulation of the Arp2/3 complex. actin polymerization ͉ immune deficiency ͉ Wiskott-Aldrich syndrome

Leukocytes rely on dynamic actindependent changes in cell shape to pass through blood vessels, which is fundamental to immune surveillance. WiskottAldrich Syndrome protein (WASp) is a hematopoietic cell-restricted cytoskeletal regulator important for modulating cell shape through Arp2/3-mediated actin polymerization. A recently identified WASp I294T mutation was shown to render WASp constitutively active in vivo, causing increased filamentous (F)-actin polymerization, high podosome turnover in macrophages, and myelodysplasia. The aim of this study was to determine the effect of WASp I294T expression in lymphocytes. Here, we report that lymphocytes isolated from a patient with WASp I294T , and in a cellular model of WASp I294T IntroductionThe Wiskott-Aldrich Syndrome protein (WASp) is a key cytoskeletal regulator in hematopoietic cells. 1 Through its multidomain structure, WASp integrates inputs from disparate signaling pathways to initiate and regulate Arp2/3-mediated actin polymerization. This is important for the normal formation of various actin-rich structures including the T-cell immune synapse, phagocytic cups, and specialized adhesion structures called podosomes in myeloid cells. [2][3][4] The majority of human disease-causing mutations in WASp are hypomorphic or null loss of function mutations, which result in Wiskott-Aldrich Syndrome (WAS). 5 Patients with WAS classically suffer from thrombocytopenia, eczema, and a broad immunodeficiency, reflecting the functional deficiency of almost all hematopoietic lineages.Recently, 3 novel human mutations (L270P, S272P, and I294T) leading to constitutive WASp activation have been reported to cause a distinct disease, X-linked neutropoenia (XLN). 6,7 All 3 are positioned in the GTPase binding domain (GBD; residues 230-288) of WASp and disrupt its autoinhibitory interaction with the Arp2/3 binding, C-terminal verprolin homology, central, acidic (VCA) domain. The result is enhanced and dysregulated WASp activity characterized by an increase in cellular filamentous (F)-actin and abnormalities of actin cytoskeletal structure and dynamics. A large kindred bearing the rare I294T mutation was recently reported, informing our understanding of the clinical spectrum of WASp I294T disease, which appears variable but is usually associated with neutropoenia and recurrent infections. 7,8 Although fatal infections have been described, infectious complications are surprisingly mild and not correlated with the degree of neutropoenia. 8 Other immunopathology is also seen, including low levels of CD4 and CD8 T cells, natural killer (NK) cell and B-cell lymphopenia, and reduced levels of IgA. To date, specific cellular effects have only been examined in myeloid cells, where defects of myelopoiesis and podosome formation have been reported. 7,9 Our aim in this report was to determine whether WASp I294T also attenuates aspects of lymphocyte function.The lymphocyte surface is dominated by microvilli: prominent actin-rich, fingerlike membrane projections involved in mediating cell-c...

IntroductionWiskott Aldrich syndrome (WAS) is a primary immunodeficiency characterized by a broad immune defect, thrombocytopenia, severe eczema, and an increased susceptibility to both autoimmunity and hematologic malignancy. 1 The disease is caused by mutations in the gene encoding the WAS protein (WASp), a key regulator of the actin cytoskeleton in hematopoietic cells. 2,3 Cytosolic WASp exists in an autoinhibited conformation promoted by constitutive binding of the EVH1 domain to WASp Interacting Protein (WIP). [4][5][6][7][8] Activation induces a conformational change exposing the VCA (Verprolin homology, Central region, Acidic domain) domain, which binds the Arp2/3 complex, 9,10 initiating actin filament nucleation and polymerization. 11 WASp activity is regulated through the combined influence of many binding partners and posttranslational modification, of which the Rho GTPase Cdc42 and tyrosine phosphorylation have particular importance. 1 Mutations resulting in complete loss of WASp expression are predictive of severe, classic WAS while missense mutations in the EVH1 domain generally result in residual low-level WASp expression and cause milder X-linked thrombocytopenia (XLT). [12][13][14][15] Despite this, there is significant clinical heterogeneity between different EVH1 missense mutations. In particular, mutations within exon 4 are associated with a more severe clinical phenotype. 14,15 A recently published multicenter analysis of the clinical outcomes of XLT patients revealed significantly increased rates of infections, autoimmunity, and malignancy resulting in a median severe-eventfree survival of only 10.2 years. 16 Therefore, further categorization and mechanistic understanding of mutations associated with more severe clinical features is important.The common mechanism of action for disease-causing EVH1 missense mutations appears to be enhanced protein degradation as a result of reduced WIP binding. Identification of the EVH1-binding surface for WIP as a long coiled surface clarified why so many different mutations could have the same effect. Based on this model, many EVH1 mutations have either been demonstrated Submitted January 13, 2012; accepted October 11, 2012. Prepublished online as Blood First Edition paper, November 15, 2012; DOI 10.1182 DOI 10. /blood-2012.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. Methods MiceC57BL/6 (B6) wild-type (Charles River Laboratories) and WAS KO mice (supplied by T. Strom, Memphis, TN) were housed in specified pathogenfree conditions and used at 6-18 weeks of age. Experiments were performed under a Home Office-approved project license (held by S.B.). Immature murine BM derived dendritic cells (BMDCs) were generated by culturing in RPMI medium supplemented with 10% FCS, 100 units/mL of penicillin, and 100 g/mL of streptomycin in...

Dendritic cells (DC) are key cells of the innate immune system required to prime adaptive immunity. Central DC functions including antigen uptake and presentation and DC migration are critically dependent on dynamic cytoskeletal reorganisation, the regulation of which remains poorly understood. Cytoskeletal studies are complicated by the fact that DC cytoarchitecture is altered considerably by maturation stimuli, including many tools employed for biological manipulation. Lentiviral vectors, capable of transducing non-dividing cells such as DC, hold promise both for experimental and therapeutic manipulation of DC gene and protein expression but controversy remains about their effect on DC maturation. Here, we have examined the potential of lentiviral vectors as tools for gene delivery to monocyte derived human DC with preservation of immature DC cytoskeletal structure and function. We show that vesicular stomatitis virus G glycoprotein (VSVG)-pseudotyped lentivectors are most efficient at transducing immature DC and their precursor monocytes. Even at high multiplicities of infection transduced DC retained an immature cytoskeletal phenotype, with no significant alteration of migration, antigen uptake or T-cell stimulation capacities. Furthermore, lentivectors did not alter subsequent functional maturation of the DC cytoskeleton in response to lipopolysaccharide exposure. Together our data show that VSVG-psudotyped lentiviral vectors are an effective tool for gene manipulation in human DC with preservation of functional immaturity and plasticity, making them ideal for studies of the DC cytoskeleton.