(Peprotech, Rocky Hill, NJ) and 10 ng/ml IL7 (Peprotech)) for 2 days. EGFP+ T cells were incubated with biotin-labeled anti- mouse CD3e mAb (2c11, BD Pharmingen) and anti-mouse CD28 mAb (37.51, eBioscience), and then conjugated with Dynabeads Biotin Binder (1:1 ratio, Invitrogen). T-microbeads conjugates were layered onto poly-L-lysine coated coverslips and incubated at 37uC for 5 min. Cells were fixed with BD Cytofix/Cytoperm (BD Biosciences) and stained with anti-ZAP-70 or anti-Lck Ab (Cell Signaling) followed by anti–rabbit Alexa555 (Molecular Probes). Z series fluorescence images were captured with a Leica DMIRB fluorescence microscope equipped with a 40x/0.55 air objective lens (Leica, Wetzlar, Germany), a Hamamatsu C4742 digital camera (Hamamatsu Photomics, Hamamatsu City, Japan) and a deconvolution system driven by Openlab software (Improvision, Lexington, MA). Deconvoluted images were pro- cessed using Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA). At least 100 cells were analyzed per condition. Subcellular fractionation Cells were lysed by brief sonication in 200 ml ice-cold buffer A (250 mM sucrose, 20 mM Tris, pH 7.8, 10 mM MgCl2, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, and complete protease inhibitor cocktail (Roche)). After centrifugation for 5 min at 900 g, took 200 ml post-nuclei (PN) fraction. PN fraction was separated into particulate membrane-containing (P100) and cytosol (C) fractions by centrifugation for 30 min at 100,0006 g. Take 200 ml supernatant as C fraction. Pellet was solubilized with 200 ml Mg2+ lysis/wash buffer (Millipore) and separated by additional 30 min at 100,0006 g. The supernatant was kept as detergent-soluble membrane (SM) fraction. The pellet was further solubilized with 200 ml 0.05% sodium dodecyl sulfate (SDS)-containing Mg2+ lysis/ wash buffer, and then centrifuged for 30 min at 13,0006 g. The supernatant was kept as detergent-insoluble membrane (IS) fraction. Equal volumes of each fraction were used for immuno- precipitation and immunoblotting. For immunoprecipitation, each fraction was 5-fold diluted with SDS-containing Mg2+ lysis/wash buffer (final SDS concentration was 0.01%). Immunoprecipitation and Lck kinase assay Wild-type and Rhoh-/- thymocytes or lymph node derived T cells were left unstimulated or stimulated anti-CD3e and CD28 mAbs for 2 min. HEK 293 cells were transfected using the CaPO4 coprecipitation procedure (Invitrogen). Jurkat T cells were transfected by electroporation (300 V, 960 mF, Bio-Rad). The expression vector for kinase-dead ZAP-70 (pCDNA3-KA-ZAP-70) and constitutively active ZAP-70 (pCDNA3-AA-ZAP-70) was kindly provided by Dr. A. Weiss[17]. The pSM-CA-Lck (constituve active Lck) and pSM-KD-Lck (kinase-dead Lck) were gifted from Dr. M.J. Bijlmakers[18]. For the transfection experiments, the following plasmids were used: HA–RhoH, pCDNA3-ZAP-70, pCDNA3-KA-ZAP-70, pCDNA3-AA-ZAP- 70, pSM-CA-Lck and pSM-KD-Lck. Cells were lysed in ice-cold Mg2+ lysis/wash buffer (Upstate Biotechnology) containing mixture of protease and phosphatase inhibitors (1 mM Na3VO4, 10 mM NaF, complete protease inhibitor cocktail). Cellular lysates (500 mg) were immunoprecipitated, using the anti-Lck (1 ml, 2752, Cell Signaling), anti-HA (100 ng, 3F10, Roche) or anti-ZAP-70 (1 mg, 1E7.2, Millipore) antibodies. The immunoprecipitated mixture or 20 mg total lysates were separated on a SDS polyacrylamide gel, and transferred to a polyvinylidene difluoride membrane (PVDF) (BioRad). The membranes were probed with antibodies indicated in each experiment. For Lck kinase assay, anti-Lck immunoprecipitate products were washed three times with lysis buffer and once with kinase buffer (50 mM Tris pH 7.5, RhoH as an Adaptor for ZAP-70 5 mM MgCl2, 5 mM MnCl2, 1 mM Na3VO4 and 1 mM DTT) and resuspended with 30 mL kinase buffer. Reaction was started by addition of 10 mCi [c-32P] ATP and left for 10 min. Reaction mixtures were subjected to SDS-PAGE and analyzed by autoradiography. Statistical Analysis Each experiment was performed two-four times. Representative data or images from one of the repeated experiments were shown in the figures. Statistical significance was determined by a Student t-Test and data with p-value less than 0.05 are considered as significant difference. Results Defective interaction between Lck and CD3f in RhoH- deficient T cells Engagement of the TCR results in the activation of the Src family phosphotyrosine kinase (PTK), Lck[19,20]. Activated Lck phos- phorylates tyrosine residues in the ITAMs in the CD3f subunits of the TCR. ZAP-70 is then recruited to the phosphorylated CD3f and is activated by Lck[19,21]. We previously reported that Rhoh-/- T cells demonstrate defective CD3f phosphorylation and ZAP-70 recruitment to the TCR complex which results in the impaired TCR signaling and T cell development[3]. We investigated whether impaired Lck activation could be responsible for this TCR signaling defect in RhoH-deficient thymocytes as well as peripheral T cells. While Lck in vitro kinase activity was similar in wild type (WT) and Rhoh-/- thymocytes (Fig. 1A), the association between Lck and CD3f and the phosphorylation of CD3f were impaired in Rhoh-/- T cells (Fig. 1A & B). Lck activity is regulated in a positive fashion by phosphorylation at tyrosine 394 (Y394)[22]. Consistent with the in vitro kinase activities noted above, the level of pY394 as demonstrated by immunoblot was comparable between WT and RhoH-deficient LN T cells (Fig. 1B). CD3f expression level was lower in Rhoh-/- thymocytes than WT (Fig. 1) due to the defective T cell development in Rhoh-/- mice with most Rhoh-/- thymocytes showing immature phenotype[3]. Published data has shown that CD3f expression is higher in single-positive thymocytes and peripheral T cells than in immature thymocytes[23]. These data suggest that while TCR signaling is abnormal, activation of Lck per se is not affected by loss of RhoH. RhoH requirement for co-recruitment of Lck and ZAP-70 to the immunologic synapse The interaction of T cells with antigen-presenting cells (APCs) results in the formation of the immunological synapse (IS). ZAP-70 has been reported to promote CD3f phosphorylation by recruiting Lck to TCR complex in a kinase activity-independent manner[9]. Both Lck and ZAP-70 have been shown to translocate to microclusters of receptors and signaling molecules in the peripheral regions of the T-APC interface during initiation of the TCR activation signal. The clusters then form a central supramolecular activation cluster (c-SMAC) of the IS[24,25,26]. Consistent with these previous observations, after conjugation with APCs we found in WT CD8+ cells that Lck translocated to the IS where it co-localized with ZAP-70 (Fig. 2A). In contrast, recruitment of both Lck and ZAP-70 to the IS was defective in Rhoh-/- T cells upon APC conjugation (Fig. 2A). This defect in IS localization was still present for up to 30 minutes after conjugation. Actin polymerization is critical for the formation of the IS by modulating the movement of molecules and lipid rafts[27,28], and is important for integrin-dependent adhesion between T cell and APC[27]. Because Rho GTPases are known to be major PLoS ONE | www.plosone.org 3 November 2010 | Volume 5 | Issue 11 | e13970