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• W2000785535 abstract "Phagocytosis is a vital first-line host defense mechanism against infection involving the ingestion and digestion of foreign materials such as bacteria by specialized cells, phagocytes. For phagocytes to ingest the foreign materials, they form an actin-based membrane structure called phagocytic cup at the plasma membranes. Formation of the phagocytic cup is impaired in phagocytes from patients with a genetic immunodeficiency disorder, Wiskott-Aldrich syndrome (WAS). The gene defective in WAS encodes Wiskott-Aldrich syndrome protein (WASP). Mutation or deletion of WASP causes impaired formation of the phagocytic cup, suggesting that WASP plays an important role in the phagocytic cup formation. However, the molecular details of its formation remain unknown. We have shown that the WASP C-terminal activity is critical for the phagocytic cup formation in macrophages. We demonstrated that WASP is phosphorylated on tyrosine 291 in macrophages, and the WASP phosphorylation is important for the phagocytic cup formation. In addition, we showed that WASP and WASP-interacting protein (WIP) form a complex at the phagocytic cup and that the WASP·WIP complex plays a critical role in the phagocytic cup formation. Our results indicate that the phosphorylation of WASP and the complex formation of WASP with WIP are the essential molecular steps for the efficient formation of the phagocytic cup in macrophages, suggesting a possible disease mechanism underlying phagocytic defects and recurrent infections in WAS patients. Phagocytosis is a vital first-line host defense mechanism against infection involving the ingestion and digestion of foreign materials such as bacteria by specialized cells, phagocytes. For phagocytes to ingest the foreign materials, they form an actin-based membrane structure called phagocytic cup at the plasma membranes. Formation of the phagocytic cup is impaired in phagocytes from patients with a genetic immunodeficiency disorder, Wiskott-Aldrich syndrome (WAS). The gene defective in WAS encodes Wiskott-Aldrich syndrome protein (WASP). Mutation or deletion of WASP causes impaired formation of the phagocytic cup, suggesting that WASP plays an important role in the phagocytic cup formation. However, the molecular details of its formation remain unknown. We have shown that the WASP C-terminal activity is critical for the phagocytic cup formation in macrophages. We demonstrated that WASP is phosphorylated on tyrosine 291 in macrophages, and the WASP phosphorylation is important for the phagocytic cup formation. In addition, we showed that WASP and WASP-interacting protein (WIP) form a complex at the phagocytic cup and that the WASP·WIP complex plays a critical role in the phagocytic cup formation. Our results indicate that the phosphorylation of WASP and the complex formation of WASP with WIP are the essential molecular steps for the efficient formation of the phagocytic cup in macrophages, suggesting a possible disease mechanism underlying phagocytic defects and recurrent infections in WAS patients. The Wiskott-Aldrich syndrome (WAS) 2The abbreviations used are: WAS, Wiskott-Aldrich syndrome; WASP, WAS protein; F-actin, filamentous actin; VCA domain, verprolin/cofilin/acidic domain; dVCA, VCA domain-deleted; Arp2/3, actin-related protein; WH1/EVH1, WASP homology1/Ena/VASP (vasodilator-stimulated phosphoprotein) homology1; WIP, WASP-interacting protein; 3-MA, 3-methyladenine; PMA, phorbol 12-myristate 13-acetate; FCS, fetal calf serum; GFP, green fluorescence protein; EGFP, enhanced GFP; siRNA, short interfering RNA; FITC, fluorescein isothiocyanate; PDZ-GEF, PDZ-guanine nucleotide exchange factor; WB domain, WASP binding domain. 2The abbreviations used are: WAS, Wiskott-Aldrich syndrome; WASP, WAS protein; F-actin, filamentous actin; VCA domain, verprolin/cofilin/acidic domain; dVCA, VCA domain-deleted; Arp2/3, actin-related protein; WH1/EVH1, WASP homology1/Ena/VASP (vasodilator-stimulated phosphoprotein) homology1; WIP, WASP-interacting protein; 3-MA, 3-methyladenine; PMA, phorbol 12-myristate 13-acetate; FCS, fetal calf serum; GFP, green fluorescence protein; EGFP, enhanced GFP; siRNA, short interfering RNA; FITC, fluorescein isothiocyanate; PDZ-GEF, PDZ-guanine nucleotide exchange factor; WB domain, WASP binding domain. is an X chromosome-linked immunodeficiency disorder. Patients with WAS suffer from severe bleeding, eczema, recurrent infections, auto-immune diseases, and an increased risk of lymphoreticular malignancy (1Wiskott A. Monatsschr. Kinderheilkd. 1937; 68: 212-216Google Scholar, 2Aldrich R.A. Steinberg A.G. Campbell D.C. Pediatrics. 1954; 13: 133-139PubMed Google Scholar, 3Ochs H.D. Thrasher A.J. J. Allergy Clin. Immunol. 2006; 117: 725-739Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). The causative gene underlying WAS encodes Wikott-Aldrich syndrome protein (WASP) (4Derry J.M. Ochs H.D. Francke U. Cell. 1994; 78 (Correction (1994) Cell79, after 922): 635-644Abstract Full Text PDF PubMed Scopus (823) Google Scholar). WASP is a 62-kDa cytosolic protein comprising distinct domains that interact with other cellular factors to regulate, mediate, and target the many functions of WASP (Fig. 3A) (5Burns S. Cory G.O. Vainchenker W. Thrasher A.J. Blood. 2004; 104: 3454-3462Crossref PubMed Scopus (119) Google Scholar, 6Barda-Saad M. Braiman A. Titerence R. Bunnell S.C. Barr V.A. Samelson L.E. Nat. Immunol. 2005; 6: 80-89Crossref PubMed Scopus (260) Google Scholar). WASP is expressed predominantly in hematopoietic cells and functions in the assembly of the actin cytoskeleton (7Higgs H.N. Pollard T.D. J. Cell Biol. 2000; 150: 1311-1320Crossref PubMed Scopus (410) Google Scholar, 8Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (557) Google Scholar), signal transduction (9Snapper S.B. Rosen F.S. Mizoguchi E. Cohen P. Khan W. Liu C.H. Hagemann T.L. Kwan S.P. Ferrini R. Davidson L. Bhan A.K. Alt F.W. Immunity. 1998; 9: 81-91Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 10Zhang J. Shehabeldin A. da Cruz L.A. Butler J. Somani A.K. McGavin M. Kozieradzki I. dos Santos A.O. Nagy A. Grinstein S. Penninger J.M. Siminovitch K.A. J. Exp. Med. 1999; 190: 1329-1342Crossref PubMed Scopus (310) Google Scholar, 11Zeng R. Cannon J.L. Abraham R.T. Way M. Billadeau D.D. Bubeck-Wardenberg J. Burkhardt J.K. J. Immunol. 2003; 171: 1360-1368Crossref PubMed Scopus (133) Google Scholar, 12Badour K. McGavin M.K. Zhang J. Freeman S. Vieira C. Filipp D. Julius M. Mills G.B. Siminovitch K.A. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1593-1598Crossref PubMed Scopus (77) Google Scholar), apoptosis (13Rawlings S.L. Crooks G.M. Bockstoce D. Barsky L.W. Parkman R. Weinberg K.I. Blood. 1999; 94: 3872-3882Crossref PubMed Google Scholar, 14Rengan R. Ochs H.D. Sweet L.I. Keil M.L. Gunning W.T. Lachant N.A. Boxer L.A. Omann G.M. Blood. 2000; 95: 1283-1292Crossref PubMed Google Scholar), and the regulation of gene expression (15Silvin C. Belisle B. Abo A. J. Biol. Chem. 2001; 276: 21450-21457Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 16Cianferoni A. Massaad M. Feske S. de la Fuente M.A. Gallego L. Ramesh N. Geha R.S. J. Allergy Clin. Immunol. 2005; 116: 1364-1371Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Furthermore, WASP translocates to lipid rafts after T-cell receptor ligation, localizing to immune synapses, the junctions between T cells, and antigen-presenting cells (17Sasahara Y. Rachid R. Byrne M.J. de la Fuente M.A. Abraham R.T. Ramesh N. Geha R.S. Mol. Cell. 2002; 10: 1269-1281Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 18Cannon J.L. Burkhardt J.K. J. Immunol. 2004; 173: 1658-1662Crossref PubMed Scopus (97) Google Scholar), and recent investigations of T-cell profiles from WASP-deficient mice have revealed a critical role for WASP in regulatory T-cell function (19Humblet-Baron S. Sather B. Anover S. Becker-Herman S. Kasprowicz D.J. Khim S. Nguyen T. Hudkins-Loya K. Alpers C.E. Ziegler S.F. Ochs H. Torgerson T. Campbell D.J. Rawlings D.J. J. Clin. Investig. 2007; 117: 407-418Crossref PubMed Scopus (151) Google Scholar, 20Marangoni F. Trifari S. Scaramuzza S. Panaroni C. Martino S. Notarangelo L.D. Baz Z. Metin A. Cattaneo F. Villa A. Aiuti A. Battaglia M. Roncarolo M.G. Dupre L. J. Exp. Med. 2007; 204: 369-380Crossref PubMed Scopus (140) Google Scholar, 21Maillard M.H. Cotta-de-Almeida V. Takeshima F. Nguyen D.D. Michetti P. Nagler C. Bhan A.K. Snapper S.B. J. Exp. Med. 2007; 204: 381-391Crossref PubMed Scopus (153) Google Scholar).The WASP C-terminal region (residues 178-502) regulates the organization of the actin cytoskeleton. Phosphatidylinositol 4,5-bisphosphate binds to the Basic Region, whereas Cdc42 binds the GTPase binding domain. Binding of these cellular factors activates the WASP C-terminal VCA (verprolin/cofilin/acidic) domain, which in turn activates the actin-related protein complex (Arp2/3 complex), stimulating actin polymerization through the formation of F-actin branch junctions (8Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (557) Google Scholar, 22Kim A.S. Kakalis L.T. Abdul-Manan N. Liu G.A. Rosen M.K. Nature. 2000; 404: 151-158Crossref PubMed Scopus (612) Google Scholar, 23Prehoda K.E. Scott J.A. Mullins R.D. Lim W.A. Science. 2000; 290: 801-806Crossref PubMed Scopus (408) Google Scholar). Recently, the Toca-1 protein (transducer of Cdc42-dependent actin assembly) was identified as a mediator of actin polymerization induced by the Cdc42·N-WASP·Arp2/3 complex (24Ho H.Y. Rohatgi R. Lebensohn A.M. Le M. Li J. Gygi S.P. Kirschner M.W. Cell. 2004; 118: 203-216Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). The WASP C terminus is also required for myoblast fusion in Drosophila (25Schafer G. Weber S. Holz A. Bogdan S. Schumacher S. Muller A. Renkawitz-Pohl R. Onel S.F. Dev. Biol. 2007; 304: 664-674Crossref PubMed Scopus (76) Google Scholar).The WASP N-terminal region (residues 1-177) comprises an N-terminal segment and the WH1/EVH1 domain. Two classes of interacting proteins have been identified; (i) the mammalian verprolins bind to the WH1/EVH1 domain, a linkage that is critical for T-cell function (17Sasahara Y. Rachid R. Byrne M.J. de la Fuente M.A. Abraham R.T. Ramesh N. Geha R.S. Mol. Cell. 2002; 10: 1269-1281Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 26Anton I.M. de la Fuente M.A. Sims T.N. Freeman S. Ramesh N. Hartwig J.H. Dustin M.L. Geha R.S. Immunity. 2002; 16: 193-204Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and monocyte chemotaxis (27Tsuboi S. J. Immunol. 2006; 176: 6576-6585Crossref PubMed Scopus (34) Google Scholar); (ii) calcium- and integrin-binding protein provides a functional bridge between WASP and integrins that plays an important role in cell adhesion by platelets (28Tsuboi S. Nonoyama S. Ochs H.D. EMBO Rep. 2006; 7: 506-511Crossref PubMed Scopus (38) Google Scholar).Verprolin was originally identified as a yeast protein implicated in cell growth, cytoskeletal organization, and endocytosis (29Vaduva G. Martin N.C. Hopper A.K. J. Cell Biol. 1997; 139: 1821-1833Crossref PubMed Scopus (105) Google Scholar). There are three mammalian homologues of verprolin that were identified through their ability to bind to the WASP N-terminal region; these are WIP (30Ramesh N. Anton I.M. Hartwig J.H. Geha R.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14671-14676Crossref PubMed Scopus (302) Google Scholar), WICH/WIRE (WIP and CR16 homologous protein/WIP related protein) (31Kato M. Miki H. Kurita S. Endo T. Nakagawa H. Miyamoto S. Takenawa T. Biochem. Biophys. Res. Commun. 2002; 291: 41-47Crossref PubMed Scopus (53) Google Scholar, 32Aspenstrom P. Exp. Cell Res. 2002; 279: 21-33Crossref PubMed Scopus (37) Google Scholar), and CR16 (33Ho H.Y. Rohatgi R. Ma L. Kirschner M.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11306-11311Crossref PubMed Scopus (77) Google Scholar). The verprolins have a common domain organization consisting of an actin binding verprolin homologous region, a glycine-rich region, a proline-rich region, and a WASP binding (WB) domain (34Anton I.M. Jones G.E. Eur. J. Cell Biol. 2006; 85: 295-304Crossref PubMed Scopus (45) Google Scholar). WIP is a widely expressed protein and is the best characterized of the mammalian verprolins. WIP plays a key role in regulating WASP, with which it appears to form a constitutive complex that functions as a unit in many cellular processes (26Anton I.M. de la Fuente M.A. Sims T.N. Freeman S. Ramesh N. Hartwig J.H. Dustin M.L. Geha R.S. Immunity. 2002; 16: 193-204Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 27Tsuboi S. J. Immunol. 2006; 176: 6576-6585Crossref PubMed Scopus (34) Google Scholar, 35Tsuboi S. J. Immunol. 2007; 178: 2987-2995Crossref PubMed Scopus (46) Google Scholar, 36Sawa M. Takenawa T. Biochem. Biophys. Res. Commun. 2006; 340: 709-717Crossref PubMed Scopus (23) Google Scholar). For example, WIP is required for targeting WASP to the immune synapse after T-cell receptor ligation (6Barda-Saad M. Braiman A. Titerence R. Bunnell S.C. Barr V.A. Samelson L.E. Nat. Immunol. 2005; 6: 80-89Crossref PubMed Scopus (260) Google Scholar, 17Sasahara Y. Rachid R. Byrne M.J. de la Fuente M.A. Abraham R.T. Ramesh N. Geha R.S. Mol. Cell. 2002; 10: 1269-1281Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). WIP expression is required for the functional expression of WASP in hematopoietic cells (37de la Fuente M.A. Sasahara Y. Calamito M. Anton I.M. Elkhal A. Gallego M.D. Suresh K. Siminovitch K. Ochs H.D. Anderson K.C. Rosen F.S. Geha R.S. Ramesh N. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 926-931Crossref PubMed Scopus (125) Google Scholar, 38Konno A. Kirby M. Anderson S.A. Schwartzberg P.L. Candotti F. Int. Immunol. 2007; 19: 185-192Crossref PubMed Scopus (35) Google Scholar), and in WIP-deficient mice, T cells fail to proliferate or polarize in response to T-cell-receptor ligation and form smaller T cell-antigen-presenting cells conjugate interfaces (26Anton I.M. de la Fuente M.A. Sims T.N. Freeman S. Ramesh N. Hartwig J.H. Dustin M.L. Geha R.S. Immunity. 2002; 16: 193-204Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). WIP also binds directly to F-actin and inhibits Ccd42-mediated actin polymerization; it synergizes with N-WASP to induce filopodia when overexpressed in fibroblasts (39Martinez-Quiles N. Rohatgi R. Anton I.M. Medina M. Saville S.P. Miki H. Yamaguchi H. Takenawa T. Hartwig J.H. Geha R.S. Ramesh N. Nat. Cell Biol. 2001; 3: 484-491Crossref PubMed Scopus (224) Google Scholar). A general role in filopodia formation is consistent with the requirement of WIP in pathogenic settings, notably the intracellular motility of vaccinia virus and Shigella, which involves the formation of an actin comet-tail mediated by the N-WASP·WIP complex (40Moreau V. Frischknecht F. Reckmann I. Vincentelli R. Rabut G. Stewart D. Way M. Nat. Cell Biol. 2000; 2: 441-448Crossref PubMed Scopus (270) Google Scholar), and the transendothelial migration of macrophages, which involves the formation of the podosomes mediated by the WASP·WIP complex (35Tsuboi S. J. Immunol. 2007; 178: 2987-2995Crossref PubMed Scopus (46) Google Scholar, 41Calle Y. Charranger N.O. Thrasher A. Jones G. J. Cell Sci. 2006; 119: 2375-2385Crossref PubMed Scopus (100) Google Scholar).Mutation or deletion of WASP causes various functional abnormalities in hematopoietic cells. In macrophages from WAS patients, WASP deficiency causes abnormal morphology (42Calle Y. Jones G.E. Jagger C. Fuller K. Blundell M.P. Chow J. Chambers T. Thrasher A.J. Blood. 2004; 103: 3552-3561Crossref PubMed Scopus (98) Google Scholar), adhesion defects (43Linder S. Nelson D. Weiss M. Aepfelbacher M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9648-9653Crossref PubMed Scopus (353) Google Scholar, 44Badolato R. Sozzani S. Malacarne F. Bresciani S. Fiorini M. Borsatti A. Albertini A. Mantovani A. Ugazio A.G. Notarangelo L.D. J. Immunol. 1998; 161: 1026-1033PubMed Google Scholar, 45Linder S. Higgs H. Hufner K. Schwarz K. Pannicke U. Aepfelbacher M. J. Immunol. 2000; 165: 221-225Crossref PubMed Scopus (128) Google Scholar, 46Zhang H. Schaff U.Y. Green C.E. Chen H. Sarantos M.R. Hu Y. Wara D. Simon S.I. Lowell C.A. Immunity. 2006; 25: 285-295Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), chemotactic defects (47Jones G.E. Zicha D. Dunn G.A. Blundell M. Thrasher A. Int. J. Biochem. Cell Biol. 2002; 34: 806-815Crossref PubMed Scopus (85) Google Scholar, 48Zicha D. Allen W.E. Brickell P.M. Kinnon C. Dunn G.A. Jones G.E. Thrasher A.J. Br. J. Haematol. 1998; 101: 659-665Crossref PubMed Scopus (201) Google Scholar), and phagocytic defects (49Lorenzi R. Brickell P.M. Katz D.R. Kinnon C. Thrasher A.J. Blood. 2000; 95: 2943-2946Crossref PubMed Google Scholar, 50Leverrier Y. Lorenzi R. Blundell M.P. Brickell P. Kinnon C. Ridley A.J. Thrasher A.J. J. Immunol. 2001; 166: 4831-4834Crossref PubMed Scopus (105) Google Scholar, 51Park J.Y. Kob M. Prodeus A.P. Rosen F.S. Shcherbina A. Remold-O'Donnell E. Clin. Exp. Immunol. 2004; 136: 104-110Crossref PubMed Scopus (70) Google Scholar, 52Linder S. Heimerl C. Fingerle V. Aepfelbacher M. Wilske B. Infect. Immun. 2001; 69: 1739-1746Crossref PubMed Scopus (49) Google Scholar).Phagocytosis involves the ingestion and digestion of foreign materials (such as microorganisms, insoluble particles, damaged or dead host cells, cell debris, and activated clotting factors (53Greenberg S. Grinstein S. Curr. Opin. Immunol. 2002; 14: 136-145Crossref PubMed Scopus (433) Google Scholar, 54Aderem A. Underhill D.M. Annu. Rev. Immunol. 1999; 17: 593-623Crossref PubMed Scopus (2048) Google Scholar, 55Underhill D.M. Ozinsky A. Annu. Rev. Immunol. 2002; 20: 825-852Crossref PubMed Scopus (832) Google Scholar)) by specialized cells called phagocytes, which include macrophages and neutrophils. Phagocytosis is, therefore, essential for host defense against infection, neoplastic proliferation, wound healing, and tissue remodeling.The phagocytic cup is an actin-based membrane structure formed at the plasma membrane of a phagocyte upon stimulation with foreign materials such as bacteria. Efficient phagocytosis requires opsonins, which are host molecules (e.g. IgG antibodies or complement proteins such as C3b) that bind to the surface of foreign materials. The opsonins are then recognized by phagocytic receptors (e.g. the Fc receptor for antibodies or CR3 for complement proteins), which induce a signal that results in polymerization of actin at the site of ingestion and the transient formation of the “phagocytic cup.” Formation of the phagocytic cup is an essential first step in phagocytosis leading to digestion of foreign materials (56Leverrier Y. Ridley A.J. Curr. Biol. 2001; 11: 195-199Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 57Olazabal I.M. Caron E. May R.C. Schilling K. Knecht D.A. Machesky L.M. Curr. Biol. 2002; 12: 1413-1418Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 58Kay J.G. Murray R.Z. Pagan J.K. Stow J.L. J. Biol. Chem. 2006; 281: 11949-11954Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Although it has been reported that WASP plays an important role in the formation of the phagocytic cup (49Lorenzi R. Brickell P.M. Katz D.R. Kinnon C. Thrasher A.J. Blood. 2000; 95: 2943-2946Crossref PubMed Google Scholar), the molecular details remain unknown.The podosome is also an actin-based structure formed at the plasma membrane of monocyte-derived cells such as macrophages, osteoclasts, and dendritic cells (76Linder S. Aephelbacher M. Trends Cell Biol. 2003; 13: 376-385Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar). Podosomes are micron-scale, dynamic, actin-rich protrusions and play essential roles in chemotactic migration and extravasation of those monocyte-derived cells (77Carman C.V. Sage P.T. Sciuto T.E. de la Fuente M.A. Geha R.S. Ochs H.D. Dvorak H.F. Dvorak A.M. Springer T.A. Immunity. 2007; 26: 784-797Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). In WASP-deficient WAS patients, the podosomes are completely absent, indicating that WASP is a critical component of the podosomes (43Linder S. Nelson D. Weiss M. Aepfelbacher M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9648-9653Crossref PubMed Scopus (353) Google Scholar). The phagocytic cup is also absent in WAS patients (49Lorenzi R. Brickell P.M. Katz D.R. Kinnon C. Thrasher A.J. Blood. 2000; 95: 2943-2946Crossref PubMed Google Scholar). These observations led us to postulate that WASP plays critical roles in the formation of both phagocytic cup and podosomes. However, molecular mechanisms of the formation of these structures including WASP are unclear. The present study focuses on understanding of the molecular basis of phagocytosis. Here we investigate how WASP regulates the formation of the phagocytic cup.EXPERIMENTAL PROCEDURESReagents—Recombinant human macrophage-colony stimulating factor-1 was purchased from R&D Systems. Anti-WASP monoclonal antibody and anti-WIP polyclonal antibody were purchased form Santa Cruz Biotechnology. Anti-phosphotyrosine monoclonal antibody (4G10) was purchased from Upstate. Anti-green fluorescence protein (GFP) monoclonal antibody (JL-8) was obtained from BD Clontech. Phenylmethylsulfonyl fluoride, leupeptin, pepstatin A, aprotinin, IGEPAL CA-630, sodium orthovanadate, anti-mouse IgG agarose, saponin, bovine serum albumin, 3-methyladenine (3-MA), latex beads (3 μm, diameter), and phorbol 12-myristate 13-acetate (PMA) were purchased form Sigma-Aldrich. RPMI1640, other tissue culture reagents, Alexa 568-labeled phalloidin, and Alexa 488-labeled secondary antibodies were obtained from Invitrogen.Cells and Transfection—Human monocyte cell line THP-1 was obtained from American Type Culture Collection and cultured in RPMI1640 containing 10% fetal calf serum (FCS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin. THP-1 cells (1-2 × 106 cells) were transfected with the WASP constructs (2 μg) using Cell Line Nucleofector Kit V and Amaxa Nucleofector (Amaxa Inc.) according to the manufacturer's instructions. After transfection, cells were cultured in 6-well plates for 2 days in RPMI1640 containing 10% FCS supplemented with 12.5 ng/ml PMA. For human primary monocyte isolation, 20-40 ml of peripheral blood was drawn from healthy volunteers after informed consent was obtained. Monocytes were isolated from blood samples using Monocyte Isolation Kit II (Miltenyi Biotech). Cells were cultured in RPMI1640 containing 10% FCS supplemented with 20 ng/ml of recombinant human macrophage-colony stimulating factor-1. Monocytes cultured for 4 days in this medium attained morphology characteristics of macrophages, and their differentiated state was confirmed by a flow cytometric analysis for CD14+ status. Monocytes cultured for 2 days were harvested and transfected with the WASP constructs using Human Monocyte Nucleofector kit and Amaxa Nucleofector (Amaxa Inc.) according to the manufacturer's instructions. After transfection, cells were cultured for 2 days additionally. Cells were co-transfected with GFP expressing plasmid, pmaxGFP (Amaxa Inc.), as a transfection marker. The efficiency of transfection measured using pmaxGFP was 40-60% for THP-1 cells or monocytes. Transfection of short interfering RNA (siRNA) was performed using DharmaFECT 2 (Dharmacon Inc.). The following sequences were chosen to generate siRNA for WASP, 5′-GCCGAGACCTCTAAACTTA-3′ (sense) and 5′-CGGCCAGATCTCAATATCAT-3′ (scrambled). The efficiency of siRNA transfection measured using fluorescein isothiocyanate (FITC)-conjugated control siRNA, BLOCK-IT (Invitrogen), was 40-60%. The Internal Review Board of Burnham Institute for Medical Research approved these experiments.Immunofluorescence Microscopy—THP-1 cells seeded on coverslips were stimulated with 12.5 ng/ml PMA for 2 days. Cells were fixed with 4% (w/v) paraformaldehyde (Fluka) for 10 min at room temperature. Fixed cells were permeabilized and blocked with phosphate-buffered saline containing 0.1% (w/v) saponin and 1% (w/v) bovine serum albumin. Cells were stained with Alexa 568-labeled phalloidin (Invitrogen) and anti-WIP or anti-WASP (Santa Cruz Biotechnology) in the presence of Fc-Block™ (BD Pharmingen). The secondary antibody, Alexa 488-labeled anti-rabbit or mouse IgG (Invitrogen), was used. Cell staining was examined under a fluorescence microscope (Zeiss Axioplan AR) or MRC 1024 SP Bio-Rad laser point scanning confocal microscope (Bio-Rad).Immunoprecipitation—For immunoprecipitation of WASP, 0.5-1 × 107 cells were lysed in buffer A (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% IGEPAL CA-630, 1 mm phenylmethylsulfonyl fluoride, 3 mm sodium orthovanadate, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin). Lysates were centrifuged at 10,000 × g at 4 °C for 15 min. The supernatant was incubated with 2 μg/ml anti-WASP monoclonal antibody (Santa Cruz Biotechnology) at 4 °C for 2 h and then incubated with anti-mouse IgG-agarose (Sigma-Aldrich) at 4 °C for 1 h. The resin binding the immune complex was washed with 0.5 ml of buffer A 3 times, and the complex was eluted with 1× Laemmli SDS-PAGE sample buffer. Eluted proteins were subjected to SDS-PAGE and analyzed by immunoblotting using anti-WASP antibody, anti-WIP polyclonal antibody (Santa Cruz Biotechnology), or anti-phosphotyrosine monoclonal antibody (4G10) (Upstate).Assay for the Phagocytic Cup Formation—Latex beads (3 μm diameter) (Sigma-Aldrich) were opsonized with 0.5 mg/ml human IgG (Sigma-Aldrich) for 16 h at room temperature, washed with phosphate-buffered saline extensively, and suspended in RPMI1640 containing 1% FCS and 10 mm 3-methyladenine (3-MA) (Sigma-Aldrich). PMA-differentiated THP-1 cells or primary macrophages grown on coverslips in 6-well culture plates were incubated in prewarmed serum-free medium containing 10 mm 3-MA for 30 min at 37 °C. Cells on coverslips were changed to 0.5 ml of ice-cold opsonized latex bead suspension at a ratio of 10-50 beads per cell and incubated for at 4 °C for 10 min to allow beads to adhere to cells. The formation of the phagocytic cups was initiated by the addition of 1.5 ml of prewarmed RPMI1640 containing 1% FCS and 10 mm 3-MA, and cells were incubated at 37 °C for a further 15 min. The phagocytic cup formation was stopped by fixation in 4% (w/v) paraformaldehyde for 10 min, and cells were permeabilized and blocked by incubating for 5 min in phosphate-buffered saline containing 0.1% (w/v) saponin and 1% (w/v) bovine serum albumin. Phagocytic cups were visualized by F-actin staining with Alexa 568-phalloidin (Invitrogen) and examined under a fluorescence microscope (Zeiss Axioplan AR).Assay for Phagocytosis—To assay phagocytosis, we measured the phagocytic uptake of IgG-opsonized fluorescence dye-conjugated latex beads (TransFluoSpheres; excitation at 488 nm/emission at 560 nm; Invitrogen) by THP-1 cells in the absence of 3-MA using a flow cytometer, FACSort (BD Biosciences). Cells were co-transfected with FITC-conjugated control siRNA and then incubated with IgG-opsonized fluorescence dye-conjugated latex beads. FITC-positive transfected cells were gated (FL1), and phagocytosis was expressed as the difference in the mean fluorescence intensity of ingested beads (FL2) measured at 37 and 4 °C.RNA Isolation and Reverse Transcription-PCR—Total RNA was isolated from THP-1 cells using PureLink 96 Total RNA Purification kit (Invitrogen) according to the manufacturer's instructions. After reverse transcription of 2 μg of total RNA by oligo(dT) priming, the resulting single strand cDNA was amplified using Expand High Fidelity PCR system (Roche Applied Science). PCR primers used were β-actin sense (5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′), β-actin antisense (5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′), WASP sense (5′-TGGACCTAGCCCAGCTGATA-3′), and WASP antisense (5′-AGGGGTCTTGTTCAGCTGA-3′). PCR was done on 100 ng of single-stranded cDNA in the presence of 5 μm each oligonucleotide primer in an Applied Biosystems 2720 Thermal Cycler (40 cycles, denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 2 min). Aliquots of 10 μl of the amplification products were separated by 1.0% agarose gel electrophoresis, visualized by ethidium bromide staining, and quantified by Alpha Imager analysis.Statistical Analysis—The significance of differences between groups was calculated by the Student's t test. Confidence (95%) was set a priori as the desired level of statistical significance.RESULTSExamination of the Phagocytic Cup Formation—When phagocytic receptors on macrophages are stimulated with opsonized foreign materials such as bacteria, phagocytic cups are transiently formed. After the macrophages completely ingest the materials, the phagocytic cups disappear (Fig. 1A). This rapid turnover of phagocytic cups makes it difficult to examine their formation. To circumvent this problem, we have developed a new experimental system. A phosphatidylinositol 3-phosphate kinase inhibitor, 3-MA, inhibits phagocytosis without affecting the formation of phagocytic cups, resulting in stabilization of the phagocytic cups (59Gagnon E. Duclos S. Rondeau C. Chevet E. Cameron P.H. Steele-Mortimer O. Paiement J. Bergeron J.J. Desjardins M. Cell. 2002; 110: 119-131Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar) (supplemental Fig. S1A). We took advantage of this effect of 3-MA on phagocytosis. To test this system, we incubated macrophages with IgG-opsonized latex beads (3-μm diameter) for 30 min. Cells were fixed, permeabilized, and stained with Alexa 568-phalloidin to visualize the phagocytic cups. Cell staining was examined under a fluorescence microscope. To quantify phagocytosis and th" @default.
• W2000785535 created "2016-06-24" @default.
• W2000785535 creator A5027783496 @default.
• W2000785535 creator A5067804353 @default.
• W2000785535 date "2007-11-01" @default.
• W2000785535 modified "2023-09-28" @default.
• W2000785535 title "Wiskott-Aldrich Syndrome Protein Is a Key Regulator of the Phagocytic Cup Formation in Macrophages" @default.
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• W2000785535 doi "https://doi.org/10.1074/jbc.m705999200" @default.
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