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The Reorientation of T-Cell Polarity and Inhibition of Immunological Synapse Formation by CD46 Involves Its Recruitment to Lipid Rafts

The Reorientation of T-Cell Polarity and Inhibition of Immunological Synapse Formation by CD46... Hindawi Publishing Corporation Journal of Lipids Volume 2011, Article ID 521863, 10 pages doi:10.1155/2011/521863 Research Article The Reorientation of T-Cell Polarity and Inhibition of Immunological Synapse Formation by CD46 Involves Its Recruitment to Lipid Rafts 1 1 1, 2 1, 2 Mandy J. Ludford-Menting, Blessing Crimeen-Irwin, Jane Oliaro, Anupama Pasam, 3, 4 1 5 6 David Williamson, Natalie Pedersen, Patricia Guillaumot, Dale Christansen, 5 3, 4 1, 2, 7 Serge Manie, Katharina Gaus, and Sarah M. Russell Peter MacCallum Cancer Centre, East Melbourne, VIC 3002, Australia Department of Surgery, University of Melbourne, Parkville, VIC 3010, Australia Centre for Vascular Research, University of New South Wales, Sydney, NSW 2052, Australia Department of Haematology, The Prince of Wales Hospital, Sydney, NSW 2052, Australia Laboratoire de G´en´etique, Signalisation et Cancer. CNRS, UMR 5201, Universit´e Claude Bernard Lyon 1, Lyon, France Department of Pathology, University of Melbourne, Parkville, VIC 3010, Australia Centre for Micro-Photonics, Swinburne University of Technology, Hawthorn, VIC 3122, Australia Correspondence should be addressed to Katharina Gaus, k.gaus@unsw.edu.au and Sarah M. Russell, sarah.russell@petermac.org Received 27 August 2010; Accepted 1 December 2010 Academic Editor: Teodor Brumeanu Copyright © 2011 Mandy J. Ludford-Menting et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Many infectious agents utilize CD46 for infection of human cells, and therapeutic applications of CD46-binding viruses are now being explored. Besides mediating internalization to enable infection, binding to CD46 can directly alter immune function. In particular, ligation of CD46 by antibodies or by measles virus can prevent activation of T cells by altering T-cell polarity and consequently preventing the formation of an immunological synapse. Here, we define a mechanism by which CD46 reorients T-cell polarity to prevent T-cell receptor signaling in response to antigen presentation. We show that CD46 associates with lipid rafts upon ligation, and that this reduces recruitment of both lipid rafts and the microtubule organizing centre to the site of receptor cross-linking. These data combined indicate that polarization of T cells towards the site of CD46 ligation prevents formation of an immunological synapse, and this is associated with the ability of CD46 to recruit lipid rafts away from the site of TCR ligation. 1. Introduction effects on immune responses [17–19]. Many of the pathogens that utilize CD46 as a receptor alter immune function in the CD46 is a human receptor for complement and many host, by both direct and indirect mechanisms [1, 2, 20, 21]. pathogens, including Neisseria, Group A Streptococcus, Immune modulation by CD46 signaling is best studied in Species B adenoviruses, vaccine strains of the measles virus, response to measles vaccine strains, where ligation of CD46 and Human Herpes Virus 6 (HHV6) [1–3]. The broad inhibits T cell activation and induces regulatory T cells range of pathogens to which CD46 can bind, combined with [22–24]. The mechanisms for this process have been difficult the ubiquitous expression of CD46, have prompted much to elucidate, in part because of the difficulty of discriminat- interest in the utility of CD46-binding viruses as oncolytic ing pleiotropic effects of the pathogen from direct effects of agents [4–11], for gene therapy [12–15], and as vectors for CD46 signaling. vaccination [16]. However, it is increasingly clear that CD46 However, recent work has identified cellular processes not only mediates entry of these infectious agents, but also that are directly affected by CD46 ligation, and that provide transmits signals upon ligation that can have important an opportunity to dissect the molecular interactions through 2 Journal of Lipids which CD46 exerts its effects. Mounting evidence suggests Final GP images were pseudocolored in Adobe Photo- that CD46 signaling affects cell morphology and polarity shop. For all images a 100 × oil objective, N = 1.4was [25, 26], and that CD46 function is regulated by intracellular used. GP histograms were fitted to two Gaussian populations compartmentalization [27, 28]. Indeed, ligation of CD46 using the nonlinear fitting algorithm Solver in Microsoft induces polarization of the T cell towards the ligation site, Excel [31]. The mean GP value of membranes adjacent to the subsequently preventing the formation of an immunological bead or of pixels masked by CD46 staining was calculated synapse, and reducing T cell signaling [25]. These obser- as described [31, 33]. Statistical analysis was performed by vations indicate that alterations in cell polarity mediated ANOVA with Tukey’s multiple comparison tests. by ligand binding to CD46 might impact upon multiple cellular functions and on immunological responses. Here, we 2.4. Isolation of Detergent Resistant Membranes (DRM). Cells establish a tractable in vitro system with which to elucidate were scraped, lysed at 3 mg proteins/ml in ice-cold TNE the mechanisms by which CD46 controls polarity, and buffer (TBS pH 7.2, 4 mM NaVO , 5 mM EGTA and protease demonstrate that the changes in polarity of T cells involve inhibitors) containing 0.5% Triton X-100 (Sigma) for 25 min a functional interaction of CD46 with lipid rafts. at 4 C and fractionated on a bottom-loaded discontinuous sucrose gradient as described [34]. Gradient fractions were 2. Materials and Methods TCA precipitated and analyzed by Western blot. For ligation, cells were incubated with anti-CD46 (1840, 20 min, 5 μg/mL, 2.1. Constructs, Cell Lines, and Reagents. CD46Cyt1.C-A 4 C) [29] and with secondary antibody (5 min, 10 μg/mL, and CD46Cyt1.C-A,L-R were generated by site-directed 37 C). mutagenesisasdescribed [27], and with CD46-Cyt1, CD46- Cyt2, and CD46-Cyt1L-R [28] expressed in the CHO-K1 cell 2.5. Palmitoylation Assay. Cells (1 × 10 )were cultured with line [29], and subcloned into pMSCV-GFP for expression in 5mCi (total) H palmitoic acid (Amersham Biosciences UK) the MD45 cell line [27]. Expression was at approximately for 4 hours at 37 Cand 5% CO ,washed once inPBS, endogenous levels (see comparison with HeLa cells in lysed in 0.5% Nonidet-P40, TBS (pH7.8), 5 mM EDTA, and Figure 1(c)). Human T cells were isolated as described [27]. Complete protease inhibitors (Roche Diagnostics, Australia) Antibodies were mouse IgG1 to Transferrin receptor (Tfr, on ice, and immunoprecipitated with anti-CD46 (1840) CD71) (BD Pharmingen, San Diego, CA); mouse IgG1 to antibody for PAGE. flotillin-2 (BD Transduction Laboratories Franklin Lakes, NJ), mouse IgG2a (E4.3) and polyclonal rabbit (1840) to CD46 [29, 30]. 3. Results 3.1. CD46 Is Recruited into Detergent-Resistant Membranes 2.2. Cell Surface Ligation and Immunofluorescent Staining. by Ligation, via a Palmitoylated Cysteine in the Transmem- MD45 cells (4 × 10 ) expressing CD46 constructs were brane Domain. Lipid rafts, defined as “small (10–200 nm), ligated with anti-CD46 (1840 rabbit polyclonal) antibody heterogeneous, highly dynamic, sterol- and sphingolipid- or anti-Tfr antibody for 30 min at 37 C, then incubated enriched domains that compartmentalize cellular processes” with antiCD3/CD28 coated beads and stained as described [35], have been implicated in T-cell polarity [36–40], so we [25]. Confocal images were acquired and processed with proposed that they might mediate CD46 effects on polarity. a BX61 microscope (Olympus, Melville, NY) and Olympus In support of the notion that CD46 might associate with Fluorview FV1000 laser-scanning confocal and software rafts, a recent study showed that CD46 was recruited to (Olympus, Japan) as described [27]. 3D images of the cells detergent-resistant membranes (DRM) after infection with were acquired with an optical distance of 0.5 μM between Human Herpes Virus 6 (HHV6) [41]. CD46 is alternatively slices. Approximately 20–30 slices were acquired per image, spliced to yield two isoforms with different cytoplasmic with the pin hole set to 1 AU, using a 60 × oil immersion domains (CD46Cyt1 and CD46Cyt2, Figure 1(a)). Each objective (NA 1.42). Digital images were processed with isoform contains a transmembrane cysteine that is a possible Image J (NIH, MD) and MetaMorph (Universal Imaging site for palmitoylation, which might enable recruitment into Corporation, PA). For MTOC recruitment, slides were de- lipid rafts [30, 42]. Multiple CD46 isoforms are expressed identified and scored for concentration of tubulin staining at in every human cell, so to determine the palmitoylation the bead interface. and raft recruitment of individual isoforms, we utilized the murine T-cell line, MD45 [27], which does not normally 2.3. Laurdan Microscopy. Cells labeled with Laurdan (Molec- express CD46 [1, 30]. First, MD45 cells expressing CD46Cyt1 ular Probes, Eugene, OR) and ligated and stimulated as were left untreated or ligated with antibody to CD46, lysed, above were excited with a 2-photon laser at 800 nm and and fractionated on a sucrose density gradient (Figure 1(b)). emission intensities simultaneously recorded in the range of The lipid raft marker flotillin (ii), but not the nonraft 400–460 nm and 470–530 nm [31, 32]. Intensity images were protein transferrin receptor (Tfr) (i), was present in the DRM converted into Generalized Polarization (GP) images (WiT (fractions 3–8). No CD46 was detected in the DRM from software) with nonligated cells (iii); however, a portion of the wild-type I − I (400–460) (470–530) CD46 was recruited into DRM after ligation (iv). These data GP = . (1) I + I (400–460) (470–530) add further support to the notion that CD46 is recruited into Journal of Lipids 3 123455789 10 Tfr (i) Flotillin (ii) CD46Cyt1 (iii) CD46Cyt1 (iv) ligated CD46Cyt1.C-A (v) ligated Short consensus repeat Transmembrane (SCR) Leader domain Cytoplasmic domain STP 1234 AB C CD46Cyt2 (vi) Variant Sequence ligated CD46Cyt1 VI CVVPYRYLQRRKKKGTYLTDETHREVKFTSL CD46Cyt2 VICVVPYRYLQRRKKKGKADGGAEYATYQTKSTTPAEQRG CD46Cyt2.C-A (vii) CD46Cyt1.C-A VI AVVPYRYLQRRKKKGTYLTDETHREVKFTS L ligated CD46Cyt2.C-A VI AVVPYRYLQRRKKKGKADGGAEYATYQTKSTTPAEQRG (a) (b) (i) (ii) H[PA] H[PA] Immunoblot (c) Figure 1: Palmitoylation regulates CD46 recruitment to DRM. (a) Schematic of CD46, with cytoplasmic sequences for the Cyt1 and Cyt2 alternatively spliced isoforms. Sequence begins within the transmembrane domain at residue 326 [28] and ends at the C-terminus. The transmembrane cysteine that we explore here is in bold and underlined. (b) MD45 cells expressing CD46 variants were either untreated (i)–(iii) or ligated with a CD46-specific antibody (iv)–(vii), lysed with TTX-100, fractionated on sucrose gradient, electrophoresed and probed with antibodies specific for Tfr, Flotillin, or CD46. Fractions 3–8 contain DRM proteins, and Fraction 10 contains the detergent- soluble protein. The lower, faint band visible in panels (iv)–(vii) represents the cross-linking antibody. (c) Cells were incubated with radiolabeled palmitoic acid, lysed and immunoprecipitated with antibodies to CD46, electrophoresed, and autoradiographed. Duplicate gels (lower panels) were immunoblotted with antibodies to CD46 to assess CD46 expression levels. (i) HeLa cells, CHO cells, and CHO cells transfected with CD46Cyt1. (ii) MD45 cells transduced with CD46Cyt1 or CD46Cyt1.C-A as shown. These data are representative of 3 independent experiments. lipid rafts upon ligation [41], providing a possible mech- To test whether the transmembrane cysteine residue of anism for the CD46-induced polarity changes. However, CD46 provides a site for palmitoylation, cells were incubated potential artefacts associated with detergent extraction [36] with radiolabeled palmitoic acid, and immunoprecipitated mean that alternative means of assessing raft association are for CD46. Radiolabeled bands of appropriate size for CD46 required to confirm this hypothesis. were detected from HeLa cells expressing endogenous HeLa CHO CD46Cyt1 CD46Cyt1 CD46Cyt1.C-A 4 Journal of Lipids CD46, and from CHO cells transfected with CD46Cyt1, the GP images (compare total GP image in column 2 with but not from nontransfected CHO cells, indicating that masked image in column 3, Figure 2(B)(i)). A high GP value CD46 is constitutively palmitoylated (Figure 1(c)(i)). In in a CD46-positive pixel gives an indication of CD46 local- transfected MD45 cells, CD46Cyt1 was also palmitoylated, isation in ordered domains that biophysically resemble lipid but mutation of the cysteine at position 328 (CD46Cyt1.C- rafts. The average GP value of CD46Cyt1 positive pixels over A) completely abolished palmitoylation (Figure 1(c)(ii)). multiple cells was 0.330 ± 0.081 (Figure 2(B)(ii), compared Similarly, CD46Cyt2 was palmitoylated, but only when with an average of 0.234 ± 0.067 for CD46Cyt1.C-A (P< the transmembrane cysteine was present (not shown). .05), indicating that palmitoylation of CD46 indeed targets Similar to CD46Cyt1, CD46Cyt2 was recruited to DRM the protein to more ordered domains. Ligated CD46Cyt1 was (Figure 1(b)(vi)), again dependent upon the cysteine residue recruited to regions of average GP values 0.437 ± 0.077 (P< (Figure 1(b)(vii)). These data combined indicate that both .001 compared with unligated CD46Cyt1, predominantly CD46 isoforms are recruited to DRM upon ligation, and red colouring in Figure 2(B)(i)(d) indicating that CD46Cyt1 that optimal recruitment requires palmitoylation of the was recruited to highly ordered raft domains after ligation. transmembrane cysteine. LigationofCD46Cyt1.C-A increased the meanGPofCD46 positive pixels (P< .05 for untreated compared with CD46 ligated), but not to the levels indicative of ordered domains (average GP 0.302 ± 0.075, predominately green colouring 3.2. CD46 Is Recruited to Ordered Membranes upon Ligation. Detergent extraction alone is not always a reliable indicator in Figure 2(B)(i)(l), P< .05 for CD46-ligated CD46Cyt1 of raft recruitment [36, 39, 40], so to test whether the compared with CD46-ligated CD46Cyt1.C-A). These studies results above represented bona fide association with lipid together demonstrate that (i) CD46 constitutively localizes rafts, we utilized a complementary approach based upon to a domain that, although not a classic raft as defined the analysis of ordered membrane structure using 2-photon by detergent insolubility or GP values, is somewhat more microscopy. The fluorescent probe, Laurdan, integrates into ordered than the general cell membranes, (ii) CD46 is all membranes but demonstrates a different emission profile recruited to stable lipid rafts upon ligation, thus increasing the abundance of ordered domains, and (iii) recruitment of if localized to the ordered domains that are considered to represent lipid rafts [31, 43, 44]. We ascribe a normalized CD46 into ordered domains (both constitutive and ligated) ratio of the two emission regions (General Polarisation is dependent upon a transmembrane cysteine. or GP) as a relative measure of membrane order, where fluid domains are arbitrarily defined as approximately 0.05– 0.25, and ordered domains are approximately 0.25–0.55 3.3. CD46 Ligation Reduces Raft Accumulation at the Site [31]. Firstly, we tested whether expression and ligation of of TCR Activation. Raft-like domains are concentrated at CD46 altered the physical properties of the plasma mem- the immunological synapse during antigen presentation [33, brane globally (Figure 2(A)). In normalized GP histograms 45–47]. In particular, TCR signaling components are asso- (Figure 2(A)(i)), we identified two populations of mem- ciated with DRM [45], and the Laurdan reporter dye branes that are characterized by the mean GP value (centre reveals condensed plasma membrane domains at T-cell of the population) and its abundance (area under the curve). activation sites [33]. We previously showed that ligation of Hence, an ordered membrane population with high mean CD46 abrogates recruitment of CD3 and the microtubule GP, “P ”, was discriminated from a broader, fluid population, organizing centre (MTOC) to the site of TCR triggering “P ”[33]. Note that the mean GP here does not reflect an [25, 48]. Because ligation of CD46 induced its association arbitrary “cut-off ” such as the 0.25 above but accommodates with ordered domains, we tested whether CD46 ligation the broad range of GP values within the population defined alters membrane condensation at T-cell activation sites by curve-fitting. Comparison of these two populations (Figure 3(a)). Activation of CD8 human T cells with beads under different conditions (Figure 2(A)(ii)) indicated that coated with antibodies to TCR components, “CD3/CD28 the proportion of condensed membranes as a percentage beads,” triggered raft formation at the cell-bead contact (GP of total membranes was 21.8% (standard deviation 2.7%) value of 0.397 ± 0.062, compare with 0.252 ± 0.087 with in parental cells, increased slightly but not significantly negative control beads coated with antibody to Tfr, P< (to 24.8%) upon transfection of CD46Cyt1, and further .05). Ligation of Tfr prior to stimulation with CD3/CD28 increased (to 34.4%, P< .05) upon ligation of CD46Cyt1. beads did not reduce membrane condensation at CD3/CD28 In contrast, transfection and ligation of CD46Cyt1.C-A had contact sites (unpublished data, P< .05). In contrast, no significant effect on global membrane order (23.4% ligation of CD46 prior to TCR stimulation significantly and 22.9% with or without ligation, P< .5compared to reduced raft formation at T cell activation sites to 0.305 ± nontransfected cells). These data indicate that expression of 0.84 (P< .001). Thus, as well as inhibiting recruitment of CD46 influences the organisation of the membrane fluidity, MTOC and cytotoxic granules to the immunological synapse and that ligation of CD46 further enhanced the formation [25], ligation of CD46 prevents lipid raft recruitment to the of ordered domains. Notably, CD46-mediated membrane site of TCR stimulation. condensation required the transmembrane cysteine. Secondly, we tested whether association of CD46 with We next determined whether CD46 was preferentially rafts was required for the inhibitory effect on raft recruitment recruited into ordered domains. To correlate CD46 with to the IS, utilizing MD45 cells (Figures 3(b) and 3(c)). As in ordered domains, we used positive CD46 staining to mask the human T cells, ordered domains were enriched at the Journal of Lipids 5 (i) CD46Cyt1 Masked DIC + CD46 GP image GP image (a) (c) (b) (i) 1.2 P : 0.220 (78.2%) (d) (f) (e) P : 0.434 (21.8%) 0.8 0.6 CD46Cyt1.C-A 0.4 (g) (i) (h) 0.2 −1 −0.5 0 0.5 1 (l) (j) (k) (ii) Mean GP (coverage) Fluid Ordered population population Cells 0.220 (78.2%) 0.434 (21.8%) (ii) CD46Cyt1 0.294 (75.2%) 0.488 (24.8%) Mean GP of CD46 +ve pixels + ligation 0.218 (65.6%) 0.468 (34.4%) Untreated CD46 ligated CD46Cyt1 0.330 ± 0.081 0.437 ± 0.077 CD46Cyt1.C-A 0.191 (76.6%) 0.440 (23.4%) CD46Cyt1.C-A 0.234 ± 0.067 0.302 ± 0.075 + ligation 0.154 (77.1%) 0.448 (22.9%) (A) (B) Figure 2: CD46 alters membrane organization and is recruited to ordered membranes. (A) The GP histograms (open squares) of ∼50 cells for each cell type (untransfected cells shown, (A)(i)) were normalized and the distribution fitted onto two Gaussian populations: a fluid population P (dotted line) and a raft-like ordered population P (dashed line). The table (A)(ii) summarizes a similar analysis of GP f o histograms in cells transfected as shown, with or without CD46 ligation. The table gives the mean GP value that was derived by fitting two populations as per the graph above. Also shown, in brackets, is the proportion of membranes associated with either of the two populations (termed coverage). (B)(i) Laurdan-labeled MD45 cells expressing CD46Cyt1 (a)–(f) or CD46Cyt1.C-A (g)–(l) were left untreated (a)–(c) and (g)–(i) or incubated with CD46-ligating antibodies (d)–(f) and (j)–(l) before fixation and imaging. Left column shows transmission images with overlaid CD46 confocal staining in red. The middle column shows pseudocolored GP images of the identical focal depth as the CD46 confocal images. GP color scale is indicated in (f) with GP values ranging from −1 (blue) to +1 (yellow). The right column shows the masked image, in which the GP values are shown only for those pixels that were defined as positive for CD46 (red coloring in (a), (d), (g), and (j)). Table (B)(ii) summarizes the GP mean ± SD of CD46-positive membranes of 40–50 images. GP values with and without ligation are significantly different (P< .05) for both CD46Cyt1 and CD46Cyt1.C-A, and CD46Cyt1 is significantly different to CD46Cyt1.C-A (P< .05), both with and without ligation. The data is representative of 3 independent experiments. site of interaction with CD3/CD28 beads (0.406 ± 0.082), (P< .05) reduction in raft recruitment to CD3/CD28 beads but expression of CD46Cyt1 reduced (0.326 ± 0.098, P< when CD46Cyt1 was expressed and not ligated, compared to .05), and ligation abrogated (0.207 ± 0.084) this recruit- parental cells that do not express CD46 or cells expressing ment.However,expression (0.388 ± 0.077) and ligation the CD46 palmitoylation mutant. This is consistent with the (0.403 ± 0.057) of CD46Cyt1.C-A had no effect. Ligated effect of CD46Cyt1 expression on global membrane organi- CD46Cyt1 colocalized with ordered domains (Figure 3(c), zation and the location of CD46Cyt1 in ordered domains, compare arrows in (iii) and (iv)), and ligated CD46Cyt1.C- suggesting that CD46-mediated raft domains are a separate A colocalized with more fluid domains (compare arrows entity to pre-existing or TCR-induced raft domains. These in (vii) and (viii)). We observed a small but significant data together indicate that CD46 must associate with lipid CD46 ligated Not ligated CD46 ligated Not ligated 6 Journal of Lipids MD45 CD46Cyt1 CD46Cyt1.C-A Untreated CD46 ligated 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 CD46 Ligated: −− + − + Beads: Tfr-Ab CD3/CD28 CD3/CD28 (a) (b) DIC + CD46 GP image DIC + CD46 GP image (i) (ii) (iii) (iv) (v) (vi) (vii) (viii) (c) Figure 3: CD46 ligation reduces raft accumulation at the immunological synapse, dependent upon the transmembrane cysteine. (a) and (b), Laurdan-labeled T cells (CD8+ human T cells (a), or CD46Cyt1 transfected MD45 cells (b)) were conjugated to beads coated with antibodies against Tfr or CD3/CD28 as indicated and activated at 37 C for 7 min, fixed and imaged. Where indicated, CD46 was first ligated for 30 min. The GP value of the membranes adjacent to the beads was measured. Each symbol represents one activation site; mean values for each condition are indicated by horizontal bars. Representative images from the CD46 transduced populations in (b) are shown in (c). Laurdan- labeled MD45 cells expressing either CD46Cyt1 (i)–(iv) or CD46Cyt1.C-A (v)–(viii) were untreated ((i), (ii), (v), (vi)) or incubated with CD46-ligating antibodies ((iii), (iv), (vii), (viii)), fixed and imaged. Panels (i), (iii), (v), and (vii) show transmission images with overlaid CD46 staining in red. Panels (ii), (iv), (vi), and (viii) show GP images with the same pseudo-coloring as in Figure 2. The GP values of the membranes adjacent to the beads in the cells shown are 0.273 (ii), 0.181 (iv), 0.374 (vi), and 0.432 (viii). Arrows in (iv) and (viii) show membrane domains that contain CD46Cyt1 and CD46Cyt1.C-A, respectively. The data is representative of 3 experiments. GP values at the site of TCR stimulation were significantly (P< .05) different between CD46-ligated and not on the TCR-stimulated primary T cells (a), and on the CD46Cyt1-transduced MD45 cells (b). rafts to prevent the polarization of rafts to the IS and that with CD46 or control antibody prior to incubation with CD46 ligation competes with TCR signalling to control the CD3/CD28 beads, and scored for MTOC polarisation to localisation of higher-ordered membranes. the beads. Transfection of the CD46 variants alone had no effect on MTOC polarisation (Figures 4(a) and 4(b),black bars, between 72% and 79% polarisation), but ligation of 3.4. An Association with Lipid Rafts Is Required, But Not CD46Cyt1 reduced MTOC recruitment to the CD3/CD28 beads (Figure 4(a), 79% to 23%, P< .001). This effect Sufficient, for the Reorientation of Polarity by CD46 Ligation. Rafts have been implicated in T cell polarity [48–50], so was specific for the CD46 antibody, because antibodies to we tested the effect of mutating the raft-association motif Tfr had no effect, and was mediated by CD46Cyt1 but not on MTOC reorientation to the CD3/CD28 beads. MD45 by CD46Cyt2 (Figure 4(a)). These data indicate that the cells expressing wild-type or mutated CD46 were treated competition for polarity between CD46 and TCR signals GP at contact site CD46Cyt1.C-A CD46Cyt1 Journal of Lipids 7 Cells alone Cyt1 Cyt2 CD46 isoform Cells alone Cyt1 Cyt1C-A Cyt1L-R Cyt1C-A, L-R CD46 isoform Untreated Tfr ligated Untreated CD46 ligated CD46 ligated (a) (b) Figure 4: Lipid raft association is necessary but not sufficient for the control of MTOC polarization by CD46 ligation. MD45 cells, untransfected or transfected with CD46 variants as shown, were untreated (black bars) or ligated with antibodies to Tfr ((a), grey bars) or CD46 ((a) and (b), white bars), then incubated with CD3/28 beads for 90 min. Cell-bead conjugates were adhered onto glass slides by centrifugation, stained for α-tubulin, and examined by immunofluorescence microscopy. Samples were blind scored for the MTOC location, given as the percentage of cells in which MTOC was found at the cell-bead interface. Error bars represent SEM for 4–8 independent experiments of 50 cells each. can be observed in MD45 cells expressing the Cyt1 isoform. 4. Discussion In contrast, ligation of CD46Cyt1.C-A in which the raft- This study shows that the competing polarizing signal binding sites were mutated caused some inhibition of T- triggered by CD46 ligation [25] is controlled in part by cell reorientation to the TCR signal (Figure 4(b), to 54%) the association of CD46 with lipid rafts. We correlate but was not as effective as ligation of wild-type CD46Cyt1 biochemical, microscopic, and functional experiments to (P< .001 for mutated CD46 compared with wild-type). The indicate the following model. Firstly, CD46 is not consti- observation that reorientation was not completely abolished tutively associated with DRM but is located in domains by mutating the raft-association site, and that CD46Cyt2 that are slightly more ordered than the average membrane. was not able to mediate reorientation, indicates that entities We observed an increase in ordered domains on trans- other than lipid rafts also play a role. Given that CD46 fection of wild-type CD46-Cyt1 but not CD46-Cyt1C-A, associates with the polarity regulator, Discs large (Dlg) via its suggesting the possibility that CD46 constitutively associates C-terminus ([28] and see below), we tested whether muta- with a subset of small, unstable rafts. Secondly, ligation of tion of the C-terminus could impact upon repolarisation. CD46 causes partial recruitment into DRM and ordered Indeed, mutation of the C-terminal residue (CD46.Cyt1L-R) significantly reduced the reorientation of the T cells, and this membrane domains (and results in an increase in abun- dance of ordered domains), indicating that ligation causes was even further reduced (but not completely abrogated) by a double mutation (CD46.Cty1C-A,L-R). These observa- membrane reorganisation and stabilization of raft domains. The palmitoylated transmembrane cysteine is critical for this tions combined indicate that the association of CD46 with recruitment, as measured both by Laurdan staining and by lipid rafts is necessary, but not sufficient, for the prevention by CD46 ligation of both raft and MTOC accumulation at detergent extraction. Thirdly, CD46-mediated reorganisa- tion of ordered membranes is partially responsible for the TCR signalling sites, and suggest that association of CD46 with Dlg might also play a role. inhibition of T-cell polarization in response to TCR signals. MTOC at site of CD3/28 ligation (%) MTOC at site of CD3/28 ligation (%) 8 Journal of Lipids Ourobservationsprovide a mechanism forour previ- indicates that rafts act in cooperation with other morpho- ous findings that competition for polarity by CD46 can logical determinants. An important conclusion from these profoundly affect the ability of T cells and NK cells to experiments is that the regulation of polarity cannot be respond to activating signals and to mediate effector func- explained by a hierarchical, stepwise contribution of the tions [25]. We find that the competition with TCR-induced different components, but rather by a consensus decision polarity is mediated by CD46Cyt1, but not CD46Cyt2. This based upon contributions from multiple components. parallels an inhibitory effect of ligation of CD46Cyt1, but not CD46Cyt2, on CD8 effector functioninvivo [24], suggesting Authors’ Contribution that competition for polarity might play an important role in these in vivo effects. In this study we ligate CD46 using K. Gaus and S. M. Russell contributed equally to the paper. antibodies, but our previous work indicates that the measles hemagglutinin can exert a similar competition for polarity Abbreviations [25], and recruitment of CD46 into DRM by HHV6 infection [41] suggests that this pathogen might also reorient polarity. Tfr: Transferrin receptor Similarities in signaling outcomes triggered by antibody DRM: Detergent resistant membranes and complement components [23] suggest that complement TTX-100: Triton X-100 might also mediate a competition for polarity. Use of the IS: Immunological synapse CD46 mutations described herein will elucidate how broad HHV-6: Herpes virus 6. a role competition for polarity might play in signaling through CD46, and whether this phenomenon is important Acknowledgments in therapeutic applications of CD46-binding pathogens. Interestingly, although these data indicate that recruit- The authors thank Amanda Gavin for initiating the palmi- ment into rafts is necessary for CD46-mediated effects on toylation experiments and Kerrie-Ann McMahon and Pat- T-cell polarity, our data with CD46. Cyt2 indicates that rick Humbert for critical comments. The work was sup- recruitment of CD46 into rafts (as measured by both DRM ported by grants from the ARC and NHMRC of Australia, the extraction and Laurdan staining) is not sufficient to prevent Wellcome Trust to SMR, and the ARC no. 4915 to S. Manie. polarisation of either rafts or the MTOC to the site of TCR stimulation. This, combined with our observation that References abrogation of raft accumulation only partially prevents re- orientation, indicates that the dominant effect of CD46 on [1] S. Russell, “CD46: a complement regulator and pathogen T-cell polarity is mediated by a combined contribution from receptor that mediates links between innate and acquired lipid rafts and other entities. We have previously found immune function,” Tissue Antigens, vol. 64, no. 2, pp. 111–118, that CD46 interacts with Dlg, a member of a network that regulates polarity of epithelial cells and T cells [27, 28, [2] R. C.Riley-Vargas, D. B. Gill, C. Kemper, M.K.Liszewski, 48], making it a likely candidate to contribute to the re- and J. P. Atkinson, “CD46: expanding beyond complement orientation mediated by CD46. The reduced re-orientation regulation,” Trends in Immunology, vol. 25, no. 9, pp. 496–503, of a mutated CD46.Cyt1 with abrogated binding to Dlg is [3] R. 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The Reorientation of T-Cell Polarity and Inhibition of Immunological Synapse Formation by CD46 Involves Its Recruitment to Lipid Rafts

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Copyright © 2011 Mandy J. Ludford-Menting et al.
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Hindawi Publishing Corporation Journal of Lipids Volume 2011, Article ID 521863, 10 pages doi:10.1155/2011/521863 Research Article The Reorientation of T-Cell Polarity and Inhibition of Immunological Synapse Formation by CD46 Involves Its Recruitment to Lipid Rafts 1 1 1, 2 1, 2 Mandy J. Ludford-Menting, Blessing Crimeen-Irwin, Jane Oliaro, Anupama Pasam, 3, 4 1 5 6 David Williamson, Natalie Pedersen, Patricia Guillaumot, Dale Christansen, 5 3, 4 1, 2, 7 Serge Manie, Katharina Gaus, and Sarah M. Russell Peter MacCallum Cancer Centre, East Melbourne, VIC 3002, Australia Department of Surgery, University of Melbourne, Parkville, VIC 3010, Australia Centre for Vascular Research, University of New South Wales, Sydney, NSW 2052, Australia Department of Haematology, The Prince of Wales Hospital, Sydney, NSW 2052, Australia Laboratoire de G´en´etique, Signalisation et Cancer. CNRS, UMR 5201, Universit´e Claude Bernard Lyon 1, Lyon, France Department of Pathology, University of Melbourne, Parkville, VIC 3010, Australia Centre for Micro-Photonics, Swinburne University of Technology, Hawthorn, VIC 3122, Australia Correspondence should be addressed to Katharina Gaus, k.gaus@unsw.edu.au and Sarah M. Russell, sarah.russell@petermac.org Received 27 August 2010; Accepted 1 December 2010 Academic Editor: Teodor Brumeanu Copyright © 2011 Mandy J. Ludford-Menting et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Many infectious agents utilize CD46 for infection of human cells, and therapeutic applications of CD46-binding viruses are now being explored. Besides mediating internalization to enable infection, binding to CD46 can directly alter immune function. In particular, ligation of CD46 by antibodies or by measles virus can prevent activation of T cells by altering T-cell polarity and consequently preventing the formation of an immunological synapse. Here, we define a mechanism by which CD46 reorients T-cell polarity to prevent T-cell receptor signaling in response to antigen presentation. We show that CD46 associates with lipid rafts upon ligation, and that this reduces recruitment of both lipid rafts and the microtubule organizing centre to the site of receptor cross-linking. These data combined indicate that polarization of T cells towards the site of CD46 ligation prevents formation of an immunological synapse, and this is associated with the ability of CD46 to recruit lipid rafts away from the site of TCR ligation. 1. Introduction effects on immune responses [17–19]. Many of the pathogens that utilize CD46 as a receptor alter immune function in the CD46 is a human receptor for complement and many host, by both direct and indirect mechanisms [1, 2, 20, 21]. pathogens, including Neisseria, Group A Streptococcus, Immune modulation by CD46 signaling is best studied in Species B adenoviruses, vaccine strains of the measles virus, response to measles vaccine strains, where ligation of CD46 and Human Herpes Virus 6 (HHV6) [1–3]. The broad inhibits T cell activation and induces regulatory T cells range of pathogens to which CD46 can bind, combined with [22–24]. The mechanisms for this process have been difficult the ubiquitous expression of CD46, have prompted much to elucidate, in part because of the difficulty of discriminat- interest in the utility of CD46-binding viruses as oncolytic ing pleiotropic effects of the pathogen from direct effects of agents [4–11], for gene therapy [12–15], and as vectors for CD46 signaling. vaccination [16]. However, it is increasingly clear that CD46 However, recent work has identified cellular processes not only mediates entry of these infectious agents, but also that are directly affected by CD46 ligation, and that provide transmits signals upon ligation that can have important an opportunity to dissect the molecular interactions through 2 Journal of Lipids which CD46 exerts its effects. Mounting evidence suggests Final GP images were pseudocolored in Adobe Photo- that CD46 signaling affects cell morphology and polarity shop. For all images a 100 × oil objective, N = 1.4was [25, 26], and that CD46 function is regulated by intracellular used. GP histograms were fitted to two Gaussian populations compartmentalization [27, 28]. Indeed, ligation of CD46 using the nonlinear fitting algorithm Solver in Microsoft induces polarization of the T cell towards the ligation site, Excel [31]. The mean GP value of membranes adjacent to the subsequently preventing the formation of an immunological bead or of pixels masked by CD46 staining was calculated synapse, and reducing T cell signaling [25]. These obser- as described [31, 33]. Statistical analysis was performed by vations indicate that alterations in cell polarity mediated ANOVA with Tukey’s multiple comparison tests. by ligand binding to CD46 might impact upon multiple cellular functions and on immunological responses. Here, we 2.4. Isolation of Detergent Resistant Membranes (DRM). Cells establish a tractable in vitro system with which to elucidate were scraped, lysed at 3 mg proteins/ml in ice-cold TNE the mechanisms by which CD46 controls polarity, and buffer (TBS pH 7.2, 4 mM NaVO , 5 mM EGTA and protease demonstrate that the changes in polarity of T cells involve inhibitors) containing 0.5% Triton X-100 (Sigma) for 25 min a functional interaction of CD46 with lipid rafts. at 4 C and fractionated on a bottom-loaded discontinuous sucrose gradient as described [34]. Gradient fractions were 2. Materials and Methods TCA precipitated and analyzed by Western blot. For ligation, cells were incubated with anti-CD46 (1840, 20 min, 5 μg/mL, 2.1. Constructs, Cell Lines, and Reagents. CD46Cyt1.C-A 4 C) [29] and with secondary antibody (5 min, 10 μg/mL, and CD46Cyt1.C-A,L-R were generated by site-directed 37 C). mutagenesisasdescribed [27], and with CD46-Cyt1, CD46- Cyt2, and CD46-Cyt1L-R [28] expressed in the CHO-K1 cell 2.5. Palmitoylation Assay. Cells (1 × 10 )were cultured with line [29], and subcloned into pMSCV-GFP for expression in 5mCi (total) H palmitoic acid (Amersham Biosciences UK) the MD45 cell line [27]. Expression was at approximately for 4 hours at 37 Cand 5% CO ,washed once inPBS, endogenous levels (see comparison with HeLa cells in lysed in 0.5% Nonidet-P40, TBS (pH7.8), 5 mM EDTA, and Figure 1(c)). Human T cells were isolated as described [27]. Complete protease inhibitors (Roche Diagnostics, Australia) Antibodies were mouse IgG1 to Transferrin receptor (Tfr, on ice, and immunoprecipitated with anti-CD46 (1840) CD71) (BD Pharmingen, San Diego, CA); mouse IgG1 to antibody for PAGE. flotillin-2 (BD Transduction Laboratories Franklin Lakes, NJ), mouse IgG2a (E4.3) and polyclonal rabbit (1840) to CD46 [29, 30]. 3. Results 3.1. CD46 Is Recruited into Detergent-Resistant Membranes 2.2. Cell Surface Ligation and Immunofluorescent Staining. by Ligation, via a Palmitoylated Cysteine in the Transmem- MD45 cells (4 × 10 ) expressing CD46 constructs were brane Domain. Lipid rafts, defined as “small (10–200 nm), ligated with anti-CD46 (1840 rabbit polyclonal) antibody heterogeneous, highly dynamic, sterol- and sphingolipid- or anti-Tfr antibody for 30 min at 37 C, then incubated enriched domains that compartmentalize cellular processes” with antiCD3/CD28 coated beads and stained as described [35], have been implicated in T-cell polarity [36–40], so we [25]. Confocal images were acquired and processed with proposed that they might mediate CD46 effects on polarity. a BX61 microscope (Olympus, Melville, NY) and Olympus In support of the notion that CD46 might associate with Fluorview FV1000 laser-scanning confocal and software rafts, a recent study showed that CD46 was recruited to (Olympus, Japan) as described [27]. 3D images of the cells detergent-resistant membranes (DRM) after infection with were acquired with an optical distance of 0.5 μM between Human Herpes Virus 6 (HHV6) [41]. CD46 is alternatively slices. Approximately 20–30 slices were acquired per image, spliced to yield two isoforms with different cytoplasmic with the pin hole set to 1 AU, using a 60 × oil immersion domains (CD46Cyt1 and CD46Cyt2, Figure 1(a)). Each objective (NA 1.42). Digital images were processed with isoform contains a transmembrane cysteine that is a possible Image J (NIH, MD) and MetaMorph (Universal Imaging site for palmitoylation, which might enable recruitment into Corporation, PA). For MTOC recruitment, slides were de- lipid rafts [30, 42]. Multiple CD46 isoforms are expressed identified and scored for concentration of tubulin staining at in every human cell, so to determine the palmitoylation the bead interface. and raft recruitment of individual isoforms, we utilized the murine T-cell line, MD45 [27], which does not normally 2.3. Laurdan Microscopy. Cells labeled with Laurdan (Molec- express CD46 [1, 30]. First, MD45 cells expressing CD46Cyt1 ular Probes, Eugene, OR) and ligated and stimulated as were left untreated or ligated with antibody to CD46, lysed, above were excited with a 2-photon laser at 800 nm and and fractionated on a sucrose density gradient (Figure 1(b)). emission intensities simultaneously recorded in the range of The lipid raft marker flotillin (ii), but not the nonraft 400–460 nm and 470–530 nm [31, 32]. Intensity images were protein transferrin receptor (Tfr) (i), was present in the DRM converted into Generalized Polarization (GP) images (WiT (fractions 3–8). No CD46 was detected in the DRM from software) with nonligated cells (iii); however, a portion of the wild-type I − I (400–460) (470–530) CD46 was recruited into DRM after ligation (iv). These data GP = . (1) I + I (400–460) (470–530) add further support to the notion that CD46 is recruited into Journal of Lipids 3 123455789 10 Tfr (i) Flotillin (ii) CD46Cyt1 (iii) CD46Cyt1 (iv) ligated CD46Cyt1.C-A (v) ligated Short consensus repeat Transmembrane (SCR) Leader domain Cytoplasmic domain STP 1234 AB C CD46Cyt2 (vi) Variant Sequence ligated CD46Cyt1 VI CVVPYRYLQRRKKKGTYLTDETHREVKFTSL CD46Cyt2 VICVVPYRYLQRRKKKGKADGGAEYATYQTKSTTPAEQRG CD46Cyt2.C-A (vii) CD46Cyt1.C-A VI AVVPYRYLQRRKKKGTYLTDETHREVKFTS L ligated CD46Cyt2.C-A VI AVVPYRYLQRRKKKGKADGGAEYATYQTKSTTPAEQRG (a) (b) (i) (ii) H[PA] H[PA] Immunoblot (c) Figure 1: Palmitoylation regulates CD46 recruitment to DRM. (a) Schematic of CD46, with cytoplasmic sequences for the Cyt1 and Cyt2 alternatively spliced isoforms. Sequence begins within the transmembrane domain at residue 326 [28] and ends at the C-terminus. The transmembrane cysteine that we explore here is in bold and underlined. (b) MD45 cells expressing CD46 variants were either untreated (i)–(iii) or ligated with a CD46-specific antibody (iv)–(vii), lysed with TTX-100, fractionated on sucrose gradient, electrophoresed and probed with antibodies specific for Tfr, Flotillin, or CD46. Fractions 3–8 contain DRM proteins, and Fraction 10 contains the detergent- soluble protein. The lower, faint band visible in panels (iv)–(vii) represents the cross-linking antibody. (c) Cells were incubated with radiolabeled palmitoic acid, lysed and immunoprecipitated with antibodies to CD46, electrophoresed, and autoradiographed. Duplicate gels (lower panels) were immunoblotted with antibodies to CD46 to assess CD46 expression levels. (i) HeLa cells, CHO cells, and CHO cells transfected with CD46Cyt1. (ii) MD45 cells transduced with CD46Cyt1 or CD46Cyt1.C-A as shown. These data are representative of 3 independent experiments. lipid rafts upon ligation [41], providing a possible mech- To test whether the transmembrane cysteine residue of anism for the CD46-induced polarity changes. However, CD46 provides a site for palmitoylation, cells were incubated potential artefacts associated with detergent extraction [36] with radiolabeled palmitoic acid, and immunoprecipitated mean that alternative means of assessing raft association are for CD46. Radiolabeled bands of appropriate size for CD46 required to confirm this hypothesis. were detected from HeLa cells expressing endogenous HeLa CHO CD46Cyt1 CD46Cyt1 CD46Cyt1.C-A 4 Journal of Lipids CD46, and from CHO cells transfected with CD46Cyt1, the GP images (compare total GP image in column 2 with but not from nontransfected CHO cells, indicating that masked image in column 3, Figure 2(B)(i)). A high GP value CD46 is constitutively palmitoylated (Figure 1(c)(i)). In in a CD46-positive pixel gives an indication of CD46 local- transfected MD45 cells, CD46Cyt1 was also palmitoylated, isation in ordered domains that biophysically resemble lipid but mutation of the cysteine at position 328 (CD46Cyt1.C- rafts. The average GP value of CD46Cyt1 positive pixels over A) completely abolished palmitoylation (Figure 1(c)(ii)). multiple cells was 0.330 ± 0.081 (Figure 2(B)(ii), compared Similarly, CD46Cyt2 was palmitoylated, but only when with an average of 0.234 ± 0.067 for CD46Cyt1.C-A (P< the transmembrane cysteine was present (not shown). .05), indicating that palmitoylation of CD46 indeed targets Similar to CD46Cyt1, CD46Cyt2 was recruited to DRM the protein to more ordered domains. Ligated CD46Cyt1 was (Figure 1(b)(vi)), again dependent upon the cysteine residue recruited to regions of average GP values 0.437 ± 0.077 (P< (Figure 1(b)(vii)). These data combined indicate that both .001 compared with unligated CD46Cyt1, predominantly CD46 isoforms are recruited to DRM upon ligation, and red colouring in Figure 2(B)(i)(d) indicating that CD46Cyt1 that optimal recruitment requires palmitoylation of the was recruited to highly ordered raft domains after ligation. transmembrane cysteine. LigationofCD46Cyt1.C-A increased the meanGPofCD46 positive pixels (P< .05 for untreated compared with CD46 ligated), but not to the levels indicative of ordered domains (average GP 0.302 ± 0.075, predominately green colouring 3.2. CD46 Is Recruited to Ordered Membranes upon Ligation. Detergent extraction alone is not always a reliable indicator in Figure 2(B)(i)(l), P< .05 for CD46-ligated CD46Cyt1 of raft recruitment [36, 39, 40], so to test whether the compared with CD46-ligated CD46Cyt1.C-A). These studies results above represented bona fide association with lipid together demonstrate that (i) CD46 constitutively localizes rafts, we utilized a complementary approach based upon to a domain that, although not a classic raft as defined the analysis of ordered membrane structure using 2-photon by detergent insolubility or GP values, is somewhat more microscopy. The fluorescent probe, Laurdan, integrates into ordered than the general cell membranes, (ii) CD46 is all membranes but demonstrates a different emission profile recruited to stable lipid rafts upon ligation, thus increasing the abundance of ordered domains, and (iii) recruitment of if localized to the ordered domains that are considered to represent lipid rafts [31, 43, 44]. We ascribe a normalized CD46 into ordered domains (both constitutive and ligated) ratio of the two emission regions (General Polarisation is dependent upon a transmembrane cysteine. or GP) as a relative measure of membrane order, where fluid domains are arbitrarily defined as approximately 0.05– 0.25, and ordered domains are approximately 0.25–0.55 3.3. CD46 Ligation Reduces Raft Accumulation at the Site [31]. Firstly, we tested whether expression and ligation of of TCR Activation. Raft-like domains are concentrated at CD46 altered the physical properties of the plasma mem- the immunological synapse during antigen presentation [33, brane globally (Figure 2(A)). In normalized GP histograms 45–47]. In particular, TCR signaling components are asso- (Figure 2(A)(i)), we identified two populations of mem- ciated with DRM [45], and the Laurdan reporter dye branes that are characterized by the mean GP value (centre reveals condensed plasma membrane domains at T-cell of the population) and its abundance (area under the curve). activation sites [33]. We previously showed that ligation of Hence, an ordered membrane population with high mean CD46 abrogates recruitment of CD3 and the microtubule GP, “P ”, was discriminated from a broader, fluid population, organizing centre (MTOC) to the site of TCR triggering “P ”[33]. Note that the mean GP here does not reflect an [25, 48]. Because ligation of CD46 induced its association arbitrary “cut-off ” such as the 0.25 above but accommodates with ordered domains, we tested whether CD46 ligation the broad range of GP values within the population defined alters membrane condensation at T-cell activation sites by curve-fitting. Comparison of these two populations (Figure 3(a)). Activation of CD8 human T cells with beads under different conditions (Figure 2(A)(ii)) indicated that coated with antibodies to TCR components, “CD3/CD28 the proportion of condensed membranes as a percentage beads,” triggered raft formation at the cell-bead contact (GP of total membranes was 21.8% (standard deviation 2.7%) value of 0.397 ± 0.062, compare with 0.252 ± 0.087 with in parental cells, increased slightly but not significantly negative control beads coated with antibody to Tfr, P< (to 24.8%) upon transfection of CD46Cyt1, and further .05). Ligation of Tfr prior to stimulation with CD3/CD28 increased (to 34.4%, P< .05) upon ligation of CD46Cyt1. beads did not reduce membrane condensation at CD3/CD28 In contrast, transfection and ligation of CD46Cyt1.C-A had contact sites (unpublished data, P< .05). In contrast, no significant effect on global membrane order (23.4% ligation of CD46 prior to TCR stimulation significantly and 22.9% with or without ligation, P< .5compared to reduced raft formation at T cell activation sites to 0.305 ± nontransfected cells). These data indicate that expression of 0.84 (P< .001). Thus, as well as inhibiting recruitment of CD46 influences the organisation of the membrane fluidity, MTOC and cytotoxic granules to the immunological synapse and that ligation of CD46 further enhanced the formation [25], ligation of CD46 prevents lipid raft recruitment to the of ordered domains. Notably, CD46-mediated membrane site of TCR stimulation. condensation required the transmembrane cysteine. Secondly, we tested whether association of CD46 with We next determined whether CD46 was preferentially rafts was required for the inhibitory effect on raft recruitment recruited into ordered domains. To correlate CD46 with to the IS, utilizing MD45 cells (Figures 3(b) and 3(c)). As in ordered domains, we used positive CD46 staining to mask the human T cells, ordered domains were enriched at the Journal of Lipids 5 (i) CD46Cyt1 Masked DIC + CD46 GP image GP image (a) (c) (b) (i) 1.2 P : 0.220 (78.2%) (d) (f) (e) P : 0.434 (21.8%) 0.8 0.6 CD46Cyt1.C-A 0.4 (g) (i) (h) 0.2 −1 −0.5 0 0.5 1 (l) (j) (k) (ii) Mean GP (coverage) Fluid Ordered population population Cells 0.220 (78.2%) 0.434 (21.8%) (ii) CD46Cyt1 0.294 (75.2%) 0.488 (24.8%) Mean GP of CD46 +ve pixels + ligation 0.218 (65.6%) 0.468 (34.4%) Untreated CD46 ligated CD46Cyt1 0.330 ± 0.081 0.437 ± 0.077 CD46Cyt1.C-A 0.191 (76.6%) 0.440 (23.4%) CD46Cyt1.C-A 0.234 ± 0.067 0.302 ± 0.075 + ligation 0.154 (77.1%) 0.448 (22.9%) (A) (B) Figure 2: CD46 alters membrane organization and is recruited to ordered membranes. (A) The GP histograms (open squares) of ∼50 cells for each cell type (untransfected cells shown, (A)(i)) were normalized and the distribution fitted onto two Gaussian populations: a fluid population P (dotted line) and a raft-like ordered population P (dashed line). The table (A)(ii) summarizes a similar analysis of GP f o histograms in cells transfected as shown, with or without CD46 ligation. The table gives the mean GP value that was derived by fitting two populations as per the graph above. Also shown, in brackets, is the proportion of membranes associated with either of the two populations (termed coverage). (B)(i) Laurdan-labeled MD45 cells expressing CD46Cyt1 (a)–(f) or CD46Cyt1.C-A (g)–(l) were left untreated (a)–(c) and (g)–(i) or incubated with CD46-ligating antibodies (d)–(f) and (j)–(l) before fixation and imaging. Left column shows transmission images with overlaid CD46 confocal staining in red. The middle column shows pseudocolored GP images of the identical focal depth as the CD46 confocal images. GP color scale is indicated in (f) with GP values ranging from −1 (blue) to +1 (yellow). The right column shows the masked image, in which the GP values are shown only for those pixels that were defined as positive for CD46 (red coloring in (a), (d), (g), and (j)). Table (B)(ii) summarizes the GP mean ± SD of CD46-positive membranes of 40–50 images. GP values with and without ligation are significantly different (P< .05) for both CD46Cyt1 and CD46Cyt1.C-A, and CD46Cyt1 is significantly different to CD46Cyt1.C-A (P< .05), both with and without ligation. The data is representative of 3 independent experiments. site of interaction with CD3/CD28 beads (0.406 ± 0.082), (P< .05) reduction in raft recruitment to CD3/CD28 beads but expression of CD46Cyt1 reduced (0.326 ± 0.098, P< when CD46Cyt1 was expressed and not ligated, compared to .05), and ligation abrogated (0.207 ± 0.084) this recruit- parental cells that do not express CD46 or cells expressing ment.However,expression (0.388 ± 0.077) and ligation the CD46 palmitoylation mutant. This is consistent with the (0.403 ± 0.057) of CD46Cyt1.C-A had no effect. Ligated effect of CD46Cyt1 expression on global membrane organi- CD46Cyt1 colocalized with ordered domains (Figure 3(c), zation and the location of CD46Cyt1 in ordered domains, compare arrows in (iii) and (iv)), and ligated CD46Cyt1.C- suggesting that CD46-mediated raft domains are a separate A colocalized with more fluid domains (compare arrows entity to pre-existing or TCR-induced raft domains. These in (vii) and (viii)). We observed a small but significant data together indicate that CD46 must associate with lipid CD46 ligated Not ligated CD46 ligated Not ligated 6 Journal of Lipids MD45 CD46Cyt1 CD46Cyt1.C-A Untreated CD46 ligated 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 CD46 Ligated: −− + − + Beads: Tfr-Ab CD3/CD28 CD3/CD28 (a) (b) DIC + CD46 GP image DIC + CD46 GP image (i) (ii) (iii) (iv) (v) (vi) (vii) (viii) (c) Figure 3: CD46 ligation reduces raft accumulation at the immunological synapse, dependent upon the transmembrane cysteine. (a) and (b), Laurdan-labeled T cells (CD8+ human T cells (a), or CD46Cyt1 transfected MD45 cells (b)) were conjugated to beads coated with antibodies against Tfr or CD3/CD28 as indicated and activated at 37 C for 7 min, fixed and imaged. Where indicated, CD46 was first ligated for 30 min. The GP value of the membranes adjacent to the beads was measured. Each symbol represents one activation site; mean values for each condition are indicated by horizontal bars. Representative images from the CD46 transduced populations in (b) are shown in (c). Laurdan- labeled MD45 cells expressing either CD46Cyt1 (i)–(iv) or CD46Cyt1.C-A (v)–(viii) were untreated ((i), (ii), (v), (vi)) or incubated with CD46-ligating antibodies ((iii), (iv), (vii), (viii)), fixed and imaged. Panels (i), (iii), (v), and (vii) show transmission images with overlaid CD46 staining in red. Panels (ii), (iv), (vi), and (viii) show GP images with the same pseudo-coloring as in Figure 2. The GP values of the membranes adjacent to the beads in the cells shown are 0.273 (ii), 0.181 (iv), 0.374 (vi), and 0.432 (viii). Arrows in (iv) and (viii) show membrane domains that contain CD46Cyt1 and CD46Cyt1.C-A, respectively. The data is representative of 3 experiments. GP values at the site of TCR stimulation were significantly (P< .05) different between CD46-ligated and not on the TCR-stimulated primary T cells (a), and on the CD46Cyt1-transduced MD45 cells (b). rafts to prevent the polarization of rafts to the IS and that with CD46 or control antibody prior to incubation with CD46 ligation competes with TCR signalling to control the CD3/CD28 beads, and scored for MTOC polarisation to localisation of higher-ordered membranes. the beads. Transfection of the CD46 variants alone had no effect on MTOC polarisation (Figures 4(a) and 4(b),black bars, between 72% and 79% polarisation), but ligation of 3.4. An Association with Lipid Rafts Is Required, But Not CD46Cyt1 reduced MTOC recruitment to the CD3/CD28 beads (Figure 4(a), 79% to 23%, P< .001). This effect Sufficient, for the Reorientation of Polarity by CD46 Ligation. Rafts have been implicated in T cell polarity [48–50], so was specific for the CD46 antibody, because antibodies to we tested the effect of mutating the raft-association motif Tfr had no effect, and was mediated by CD46Cyt1 but not on MTOC reorientation to the CD3/CD28 beads. MD45 by CD46Cyt2 (Figure 4(a)). These data indicate that the cells expressing wild-type or mutated CD46 were treated competition for polarity between CD46 and TCR signals GP at contact site CD46Cyt1.C-A CD46Cyt1 Journal of Lipids 7 Cells alone Cyt1 Cyt2 CD46 isoform Cells alone Cyt1 Cyt1C-A Cyt1L-R Cyt1C-A, L-R CD46 isoform Untreated Tfr ligated Untreated CD46 ligated CD46 ligated (a) (b) Figure 4: Lipid raft association is necessary but not sufficient for the control of MTOC polarization by CD46 ligation. MD45 cells, untransfected or transfected with CD46 variants as shown, were untreated (black bars) or ligated with antibodies to Tfr ((a), grey bars) or CD46 ((a) and (b), white bars), then incubated with CD3/28 beads for 90 min. Cell-bead conjugates were adhered onto glass slides by centrifugation, stained for α-tubulin, and examined by immunofluorescence microscopy. Samples were blind scored for the MTOC location, given as the percentage of cells in which MTOC was found at the cell-bead interface. Error bars represent SEM for 4–8 independent experiments of 50 cells each. can be observed in MD45 cells expressing the Cyt1 isoform. 4. Discussion In contrast, ligation of CD46Cyt1.C-A in which the raft- This study shows that the competing polarizing signal binding sites were mutated caused some inhibition of T- triggered by CD46 ligation [25] is controlled in part by cell reorientation to the TCR signal (Figure 4(b), to 54%) the association of CD46 with lipid rafts. We correlate but was not as effective as ligation of wild-type CD46Cyt1 biochemical, microscopic, and functional experiments to (P< .001 for mutated CD46 compared with wild-type). The indicate the following model. Firstly, CD46 is not consti- observation that reorientation was not completely abolished tutively associated with DRM but is located in domains by mutating the raft-association site, and that CD46Cyt2 that are slightly more ordered than the average membrane. was not able to mediate reorientation, indicates that entities We observed an increase in ordered domains on trans- other than lipid rafts also play a role. Given that CD46 fection of wild-type CD46-Cyt1 but not CD46-Cyt1C-A, associates with the polarity regulator, Discs large (Dlg) via its suggesting the possibility that CD46 constitutively associates C-terminus ([28] and see below), we tested whether muta- with a subset of small, unstable rafts. Secondly, ligation of tion of the C-terminus could impact upon repolarisation. CD46 causes partial recruitment into DRM and ordered Indeed, mutation of the C-terminal residue (CD46.Cyt1L-R) significantly reduced the reorientation of the T cells, and this membrane domains (and results in an increase in abun- dance of ordered domains), indicating that ligation causes was even further reduced (but not completely abrogated) by a double mutation (CD46.Cty1C-A,L-R). These observa- membrane reorganisation and stabilization of raft domains. The palmitoylated transmembrane cysteine is critical for this tions combined indicate that the association of CD46 with recruitment, as measured both by Laurdan staining and by lipid rafts is necessary, but not sufficient, for the prevention by CD46 ligation of both raft and MTOC accumulation at detergent extraction. Thirdly, CD46-mediated reorganisa- tion of ordered membranes is partially responsible for the TCR signalling sites, and suggest that association of CD46 with Dlg might also play a role. inhibition of T-cell polarization in response to TCR signals. MTOC at site of CD3/28 ligation (%) MTOC at site of CD3/28 ligation (%) 8 Journal of Lipids Ourobservationsprovide a mechanism forour previ- indicates that rafts act in cooperation with other morpho- ous findings that competition for polarity by CD46 can logical determinants. An important conclusion from these profoundly affect the ability of T cells and NK cells to experiments is that the regulation of polarity cannot be respond to activating signals and to mediate effector func- explained by a hierarchical, stepwise contribution of the tions [25]. We find that the competition with TCR-induced different components, but rather by a consensus decision polarity is mediated by CD46Cyt1, but not CD46Cyt2. This based upon contributions from multiple components. parallels an inhibitory effect of ligation of CD46Cyt1, but not CD46Cyt2, on CD8 effector functioninvivo [24], suggesting Authors’ Contribution that competition for polarity might play an important role in these in vivo effects. In this study we ligate CD46 using K. Gaus and S. M. Russell contributed equally to the paper. antibodies, but our previous work indicates that the measles hemagglutinin can exert a similar competition for polarity Abbreviations [25], and recruitment of CD46 into DRM by HHV6 infection [41] suggests that this pathogen might also reorient polarity. Tfr: Transferrin receptor Similarities in signaling outcomes triggered by antibody DRM: Detergent resistant membranes and complement components [23] suggest that complement TTX-100: Triton X-100 might also mediate a competition for polarity. Use of the IS: Immunological synapse CD46 mutations described herein will elucidate how broad HHV-6: Herpes virus 6. a role competition for polarity might play in signaling through CD46, and whether this phenomenon is important Acknowledgments in therapeutic applications of CD46-binding pathogens. Interestingly, although these data indicate that recruit- The authors thank Amanda Gavin for initiating the palmi- ment into rafts is necessary for CD46-mediated effects on toylation experiments and Kerrie-Ann McMahon and Pat- T-cell polarity, our data with CD46. Cyt2 indicates that rick Humbert for critical comments. The work was sup- recruitment of CD46 into rafts (as measured by both DRM ported by grants from the ARC and NHMRC of Australia, the extraction and Laurdan staining) is not sufficient to prevent Wellcome Trust to SMR, and the ARC no. 4915 to S. Manie. polarisation of either rafts or the MTOC to the site of TCR stimulation. 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