Moesin Interacts with the Cytoplasmic Region of Intercellular Adhesion Molecule-3 and Is Redistributed to the Uropod of T Lymphocytes During Cell Polarization Juan M
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Published September 22, 1997 Moesin Interacts with the Cytoplasmic Region of Intercellular Adhesion Molecule-3 and Is Redistributed to the Uropod of T Lymphocytes during Cell Polarization Juan M. Serrador,* José L. Alonso-Lebrero,* Miguel A. del Pozo,* Heinz Furthmayr,‡ Reinhard Schwartz-Albiez,§ Javier Calvo,i Francisco Lozano,i and Francisco Sánchez-Madrid* Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, 28006 Madrid, Spain; ‡Department of Pathology, Stanford University, Stanford, California 94305-5324; §Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany D-69120; and iServei Inmunología, Hospital Clinic, 08036 Barcelona, Spain Downloaded from Abstract. During activation, T lymphocytes become inducing anti–ICAM-3 HP2/19 mAb, as well as in the motile cells, switching from a spherical to a polarized constitutively polarized T cell line HSB-2. In addition, shape. Chemokines and other chemotactic cytokines in- moesin is associated with CD44, but not with ICAM-1, duce lymphocyte polarization with the formation of a in polarized T lymphocytes. A correlation between the uropod in the rear pole, where the adhesion receptors degree of moesin–ICAM-3 interaction and cell polar- intercellular adhesion molecule-1 (ICAM-1), ICAM-3, ization was found as determined by immunofluores- and CD44 redistribute. We have investigated mem- cence and immunoprecipitation analysis done in paral- jcb.rupress.org brane–cytoskeleton interactions that play a key role in lel. The moesin–ICAM-3 interaction was specifically the redistribution of adhesion receptors to the uropod. mediated by the cytoplasmic domain of ICAM-3 as re- Immunofluorescence analysis showed that the ERM vealed by precipitation of moesin with a GST fusion proteins radixin and moesin localized to the uropod of protein containing the ICAM-3 cytoplasmic tail from human T lymphoblasts treated with the chemokine metabolically labeled Jurkat T cell lysates. The interac- RANTES (regulated on activation, normal T cell ex- tion of moesin with ICAM-3 was greatly diminished on May 9, 2012 pressed, and secreted), a polarization-inducing agent; when RANTES-stimulated T lymphoblasts were pre- radixin colocalized with arrays of myosin II at the neck treated with the myosin-disrupting drug butanedione of the uropods, whereas moesin decorated the most dis- monoxime, which prevents lymphocyte polarization. tal part of the uropod and colocalized with ICAM-1, Altogether, these data indicate that moesin interacts ICAM-3, and CD44 molecules. Two other cytoskeletal with ICAM-3 and CD44 adhesion molecules in uropods proteins, b-actin and a-tubulin, clustered at the cell of polarized T cells; these data also suggest that these leading edge and uropod, respectively, of polarized interactions participate in the formation of links be- lymphocytes. Biochemical analysis showed that moesin tween membrane receptors and the cytoskeleton, coimmunoprecipitates with ICAM-3 in T lymphoblasts thereby regulating morphological changes during cell stimulated with either RANTES or the polarization- locomotion. ctivated T lymphocytes are motile cells with a high membrane, cytoskeletal redistribution, and polarized se- degree of asymmetry. They can emigrate to in- cretion of cytokines, have been described in T cells during A flammatory sites in response to chemoattractant cell–cell interactions (Kupfer et al., 1986, 1991; Kupfer et gradients and are able to interact with antigen-presenting al., 1994). Motile T cells exhibit an inherent polarity be- and target cells (Crabtree and Clipstone, 1994). Different fore contact with other cells, showing a zone of high sensi- aspects of cell polarization, such as modification of plasma tivity to antigen and chemokines at the leading edge (Neg- ulescu et al., 1996; Nieto et al., 1997), and the formation of a membrane protrusion, termed uropod, at the opposite Address all correspondence to F. Sánchez-Madrid, Servicio de Immu- nología, Hospital de la Princesa, universidad Autónoma de Madrid, 28006 pole of cell locomotion. It has recently been observed that Madrid, Spain. Tel.: 34-1-4023347. Fax: 34-1-3092496. E-mail: fsmadrid/ the lymphocyte uropod extends from the area of adhesion [email protected] towards the outer milieu, and that several adhesion mole- The Rockefeller University Press, 0021-9525/97/09/1409/15 $2.00 The Journal of Cell Biology, Volume 138, Number 6, September 22, 1997 1409–1423 http://www.jcb.org 1409 Published September 22, 1997 cules (intercellular adhesion molecules [ICAMs],1 CD44 the intracellular region of ICAM-3, and it correlated with hyaluronic receptor, and CD43) cluster in this structure the degree of cell polarity. These data suggest that moesin (del Pozo et al., 1995). Lymphocyte polarization with uro- is important for the redistribution of adhesion molecules pod formation is induced by several chemokines (del Pozo to the cellular uropod. et al., 1995) and other chemotactic cytokines such as inter- leukin-2 (IL-2) and IL-15 (Wilkinson and Liew, 1995; Ni- Materials and Methods eto et al., 1996), as well as by some specific polarization- inducing ICAM-3 and CD43 mAb (Campanero et al., Antibodies, Chemokines, and Reagents 1994; Sánchez-Mateos et al., 1995). In this regard, it has re- cently been found that the chemokine-induced redistribu- The anti–ICAM-3 HP2/19 and TP1/25, anti–VLA-4 HP2/1, anti-CD44 tion of adhesion receptors ICAM-1 and -3 to the uropod HP2/9, and anti-moesin/radixin 38/37 mAbs have been described (Cam- panero et al., 1991, 1993; Pulido et al., 1991; Lankes et al., 1988). The anti– plays an important role in the recruitment of other lym- ICAM-1 Hu5/3 mAb was kindly provided by Dr. F.W. Luscinskas (Har- phocytes to inflammatory foci (del Pozo et al., 1997). vard Medical School, Boston, MA). The moesin-specific polyclonal antise- Membrane interactions with the cytoskeleton appear to rum (pAb) 95/2 was raised in rabbits by immunization with recombinant be necessary in general for the formation of specialized human moesin and purified by affinity chromatography (Amieva and Furthmayr, 1995). The affinity-purified polyclonal antibodies 464, 457, protrusions. Since very few integral membrane proteins and 454 raised against unique peptides from murine ezrin, radixin, and have been found to interact directly with actin, it is likely moesin, respectively (Winckler et al., 1994), as well as the 13/H9 mAb, that accessory proteins serve to connect the actin cytoskel- which recognizes all three ERM members (Birgbauer and Solomon, eton with the plasma membrane (Hitt and Luna, 1994). 1989), were generously provided by Dr. F. Solomon (Department of Biol- Possible candidates for this role are the closely related ogy and Center for Cancer Research, Massachusetts Institute of Technol- ogy, Cambridge, MA). The anti–b-actin mAb, anti–a-tubulin mAb, and ezrin, radixin, and moesin proteins (ERM) (Tsukita et al., rabbit anti-myosin polyclonal antibodies were purchased from Sigma 1992, 1997; Bretscher, 1993; Arpin et al., 1994). Thus, pre- Chemical Co. (St. Louis, MO). P3X63 myeloma protein (IgG1, Kappa) vious studies have demonstrated that CD43 in thymocytes was used as negative control. Recombinant human moesin was obtained Downloaded from and CD44 in fibroblasts are associated with one or more of as described (Lankes and Furthmayr, 1991). Recombinant human (rh) RANTES (regulated on activation, normal T cell expressed, and secreted) these proteins (Yonemura et al., 1993; Tsukita et al., (specific activity 2–5 3 103 U/mg, purity .97%, endotoxin level ,0.1 ng/ 1994). The CD44 interaction is regulated by the small G mg cytokine) and recombinant human monocyte chemotactic protein protein Rho and phosphoinositides (Hirao et al., 1996). (rhMCP-1) (purity .99%) were purchased from R&D systems (Minneap- Ezrin, radixin, and moesin are variably associated with cell olis, MN). Butanedione monoxime was purchased from Sigma Chemical surface protrusions, such as microvilli, filopodia, micro- Co. The 80-kD fibronectin fragment (FN80) was a generous gift of Dr. A. García Pardo (Centro de Investigaciones Biológicas, Madrid, Spain). spikes, adhesion contacts, and membrane ruffling (Sato et jcb.rupress.org al., 1992; Amieva and Furthmayr, 1995). Ezrin has been Cells detected mainly in microvilli of brush border epithelial cells, such as intestinal or placental microvilli (Berryman Resting peripheral blood lymphocytes were isolated from fresh human blood by Ficoll Hypaque density gradient centrifugation (Pharmacia Bio- et al., 1995; Fath and Burgess, 1995), whereas radixin is lo- tech. Sverige, Uppsala, Sweden), followed by adherence incubation on calized in adherens junctions of epithelial cells (Tsukita et plastic flasks. Human T lymphoblasts were prepared from peripheral al., 1989). The third member of this small protein family, blood mononuclear cells by treatment with phytohemagglutinin 0.5% on May 9, 2012 membrane-organizing extension spike protein (moesin), is (Pharmacia Biotech. Sverige) for 48 h. Cells were washed and cultured in RPMI 1640 (Flow Laboratories, Irvine, Scotland) containing 10% FCS a 78-kD protein characterized initially as a heparin-bind- (Flow Laboratories) and 50 U/ml IL-2 kindly provided by Eurocetus ing protein (Lankes et al., 1988; Lankes and Furthmayr, (Madrid, Spain). T lymphoblasts cultured by 10–15 d were used in all ex- 1991). Moesin is strongly expressed in lymphoid, endothe- periments. These cells were analyzed by flow cytometry, and their pheno- lial, and several malignant cells types.