Intracytoplasmic domains of MHC class II molecules are essential for lipid-raft-dependent signaling

doi:10.1242/jcs.00449

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doi: 10.1242/10.1242/jcs.00449

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Intracytoplasmic domains of MHC class II molecules are essential for lipid-raft-dependent signaling

Stéphane Bécart^*^,1, Niclas Setterblad^*^,1, Suzanne Ostrand-Rosenberg², Santa J. Ono³, Dominique Charron¹ and Nuala Mooney¹^,

^¹ Unité INSERM U 396, Institut Biomédical des Cordeliers, 15 rue de l'Ecole de Médecine, 75006 Paris, France
^² Department of Biological Sciences, University of Maryland, Baltimore, MD 21250, USA
^³ Department of Immunology, Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK

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Fig. 4. I-A^k mediated actin reorganization is DIG dependent and requires the intracytoplasmic domains. (A) SaI/A^k and SaI/A^k tr cells were stimulated with 10.2.16 mAb-coated beads (Anti-I-A^k) or irrelevant mouse Ig-coated beads (nonstimulated, NS) at a ratio of one bead per cell for 30 minutes. Cells were cytospun, fixed and permeabilized. Polymerized actin was detected with Alexa488-phalloidin. The bright-field images of single cell/bead conjugates (a,c,e,g,i,k) and the corresponding fluorescent image of the actin cytoskeleton are presented (b,d,f,h,j,l). Polarization of the actin network was observed only at the site of SaI/A^k cell/10.2.16-coated bead contact (d) and was prevented by pre-treatment with MßCD (f). By contrast, no reorganization of the actin cytoskeleton was detected after engagement of SaI/A^k tr cells with 10.2.16-coated beads (j). (B) The percentage of cells undergoing actin reorganization was determined. At least 200 cell-bead interactions from each condition were scored and cells in which actin relocalization had occurred (to at least the same degree as shown in A, panel d) were counted as positive events. Data from one of three typical experiments is shown.

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Fig. 6. Truncation of I-A^k prevents DIG-dependent PKC- ${alpha}$ translocation to the site of I-A^k engagement. (A) SaI/A^k and SaI/A^k tr cells were incubated with 10.2.16-coated beads (Anti-I-A^k) or irrelevant mouse Ig-coated beads (nonstimulated, NS) for 30 minutes. Cells were cytospun, fixed and stained with anti-PKC- ${alpha}$ antibody and revealed with a FITC-labeled secondary antibody. Bright-field images (a,c,e,g,i,k) indicate the site of interaction between the bead and the cell and the corresponding fluorescent images reveal the localization of PKC- ${alpha}$ (b,d,f,h,j,l). A single cell-bead conjugate is shown in each panel. Cells pretreated with MßCD prior to I-A^k stimulation (Anti-I-A^k + MßCD) are indicated. (B) The percentage of cells undergoing PKC- ${alpha}$ recruitment to the site of I-A^k engagement was determined. At least 200 cell-bead interactions from each condition were examined and cells in which PKC- ${alpha}$ recruitment had occurred (to at least the same degree as shown in A, panel d) were counted as positive events. Data from one of three typical experiments is shown.

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Fig. 1. Analysis of MHC class II molecules in SaI/A^k cells. (A) Surface expression of I-A^k molecules on SaI, SaI/A^k and SaI/A^k tr cells was determined by FACS analysis. Binding of I-A^k mAb and the isotype control Ig (IgG_2b) is shown. (B) Peptide-associated MHC class II ${alpha}$ ß compact heterodimers (C ${alpha}$ ß), high-molecular-weight (HMW) complexes and free ß chains were detected by western blotting on total cell lysates from SaI, SaI/A^k and SaI/A^k tr cells. Lysates were incubated in Laemmli sample buffer for 30 minutes at room temperature (nb, lanes 1, 2) or for 10 minutes at 95°C (b, lanes 3 and 5). Samples were migrated on a 10% SDS-PAGE gel before transfer to a PVDF membrane and immunoblotting with 10.2.16 mAb.

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Fig. 2. DIGs are characterized by their GM1 and caveolin content. DIGs were characterized in SaI/A^k cells by examining the localization of the typically DIG residents glycolipid GM1 and caveolin compared with the typically non-DIG-localizing protein ß-tubulin in the sucrose gradient. Cell lysates were fractionated on sucrose gradient and all fractions were immunoblotted with the respective Abs. DIG fractions correspond to fractions 3 and 4, whereas the soluble fractions correspond to fractions 7 and 8.

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Fig. 3. MHC class II engagement induces the recruitment of I-A^k and I-A^k tr molecules into DIGs. The distribution of I-A^k and I-A^k tr molecules through the sucrose gradient and its modulation in response to I-A^k stimulation was analyzed by western blotting. (A) SaI/A^k cells were pretreated or not with MßCD for 15 minutes, and stimulated or not (nonstimulated, NS) via I-A^k for 15 minutes before lysis and fractionation on sucrose gradient. Fractions were incubated in Laemmli buffer for 30 minutes at room temperature (non-boiled) or for 10 minutes at 95°C (boiled). Samples were migrated on 10% SDS-PAGE gels, transferred to PVDF and immunoblotted with 10.2.16 mAb. C ${alpha}$ ß, compact ${alpha}$ ß dimers of MHC class II molecules; HMW, high-molecular-weight complexes; ß chain, free ß chains. (B) SaI/A^k tr cells were stimulated or not via I-A^k, lysed and fractionated as above, followed by immunoblotting with 10.2.16 mAb. (C) Immunoblots of free ß chains (boiled condition) of I-A^k and I-A^k tr were scanned and each fraction was quantified by densitometry. The results are expressed as percentage of DIG-associated MHC class II. Representative data from one of three typical experiments is shown.

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Fig. 5. I-A^k-mediated signaling in SaI/A^k cells induces recruitment of PKC- ${alpha}$ into DIGs. (A) Soluble and DIG fractions from SaI/A^k and SaI/A^k tr cells, stimulated or not via I-A^k for 15 minutes, were immunoblotted with anti-PKC- ${alpha}$ , - ${delta}$ , -µ Abs. Only the PKC- ${alpha}$ isoenzyme was detected in DIGs isolated from either SaI/A^k or SaI/A^k tr. (B) Cholesterol dependence of PKC- ${alpha}$ recruitment was analyzed in SaI/A^k cells pretreated or not with MßCD for 15 minutes and stimulated or not via I-A^k for 15 minutes. Soluble and DIG fractions were immunoblotted (IB) with anti-PKC- ${alpha}$ Ab. Pretreatment of SaI/A^k with MßCD completely abrogated PKC- ${alpha}$ recruitment to DIGs.

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Fig. 7. PKC phosphorylation and time course of recruitment into DIGs. (A) SaI/A^k and SaI/A^k tr cells were stimulated or not via I-A^k for 15 minutes, lysed and fractionated on a sucrose gradient. DIG fractions were migrated on SDS-PAGE, transferred on PVDF and immunoblotted with the specific phospho-PKC Ab. (B) Kinetics of I-A^k recruitment, PKC- ${alpha}$ and phospho-PKC recruitment to DIGs were analyzed in SaI/A^k cells. SaI/A^k cells were stimulated via I-A^k for the indicated times, lysed and fractionated on a sucrose gradient. DIG and soluble fractions were immunoblotted with the corresponding Abs for PKC- ${alpha}$ and phospho-PKC, and western blots were analyzed by densitometric quantification. Time point 0 is arbitrarily set to 1 in both experiments. The time course of I-A^k recruitment to DIGs was determined by SDS-PAGE under boiled conditions followed by 10.2.16 mAb immunoblotting.

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Fig. 8. Recruitment of MHC class II molecules to DIGs is independent of MHC class II-mediated signaling. (A) SaI/A^k cells were pretreated with the inhibitor of actin polymerization cytochalasin D (Cyt.D), or with the PKC inhibitor calphostin C (Calph.C) prior to engagement of I-A^k for 15 minutes at 37°C. I-A^k recruitment to the DIG-containing fraction isolated from SaI/A^k was unchanged in the presence of the above inhibitors as detected by immunoblotting. (B) The effect of cytochalasin D on actin reorganization induced by I-A^k engagement was revealed by confocal microscopy of cell-bead conjugates. Panels a and b represent, respectively, bright-field imaging and Alexa488-phalloidin staining of SaI/A^k cells stimulated with 10.2.16-coated beads. Panels c and d represent, respectively, bright-field imaging and Alexa488-phalloidin staining of SaI/A^k cells pretreated with Cyt.D prior to 10.2.16-coated beads stimulation. (C) The effect of calphostin C on PKC- ${alpha}$ recruitment induced by I-A^k engagement was revealed by confocal microscopy of cell-bead conjugates. Panels a and b represent, respectively, bright-field imaging and PKC- ${alpha}$ staining of SaI/A^k cells stimulated with 10.2.16-coated beads. Panels c and d represent, respectively, bright-field imaging and PKC- ${alpha}$ staining of SaI/A^k cells pretreated with Calph.C prior to 10.2.16-coated beads stimulation.

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