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The involvement of lipid rafts in the regulation of integrin function

¹ Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, London, WC2A 3PX, UK
² Sackler Institute for Muscular Skeletal Research, Department of Medicine, University College London, 5 University Street, London, WC1E 6JJ, UK

View larger version (46K): [in a new window]   Fig. 1. Integrin LFA-1 has higher affinity for lipid raft patches on J-ß2.7 cells expressing I-LFA-1 than on J-ß2.7 cells expressing wt LFA-1. Cells were incubated with TRITC-conjugated Ctx-B, then crosslinked with rabbit anti-Ctx-B antibody. Cells were fixed and stained with mAbs against LFA-1 (A), DAF (B), or TfR (C), followed by Alexa 488-conjugated goat anti-mouse IgG. Single optical sections taken at mid-height of the cells are shown. Top panels, I-LFA-1-expressing cells; bottom panels, wt LFA-1-expressing cells. Cell surface proteins, green; Ctx-B, red. Data are representative of six experiments (A), and three experiments (B,C). Bars, 10 µm.

View larger version (15K): [in a new window]   Fig. 2. Colocalisation of LFA-1, DAF and TfR with lipid raft patches. Experimental details are as described in Fig. 1. Patches of the different cell surface markers were scored into three categories: good (>80% overlap); medium (partial but clearly overlapping regions); and none (random distribution or segregation of staining). The percentages of cells falling into each category are expressed as means±s.d. Data are representative of six experiments for LFA-1 (A), and three experiments for each of DAF and TfR (B,C). Black bars, J-ß2.7 cells expressing I-LFA-1; white bars, J-ß2.7 cells expressing wt LFA-1.

View larger version (44K): [in a new window]   Fig. 3. Integrin-activating agonists cause wt LFA-1 to move into lipid rafts. Cells were incubated with no agonist (-), 0.5 mM Mn2+ (Mn2+), or 100 nM PdBu (PdBu) for 30 minutes at 37°C. (A) Adhesion to ICAM-1. Cells were allowed to bind to plastic-coated ICAM-1 (with or without stimulation) before washing and quantitation of bound cells. (B) Colocalisation of wt LFA-1 with lipid raft patches. After incubation with or without agonists, cells were incubated with TRITC-conjugated Ctx-B, then crosslinked with rabbit anti-Ctx antibody. Cells were fixed and stained with mAbs against LFA-1, followed by Alexa 488-conjugated goat anti-mouse IgG. Data are representative of three experiments. Bar, 10 µm. (C) Colocalisation of LFA-1 with lipid raft patches. Patches of LFA-1 staining were scored into three categories: good, medium, or no colocalisation (Fig. 2). The percentages of cells falling into each category are expressed as averages±s.d. Data are representative of three experiments. Black bars, unstimulated cells; white bars, cells incubated with Mn2+; grey bars, cells incubated with PdBu.

View larger version (37K): [in a new window]   Fig. 4. Integrin 4ß1 has higher affinity for lipid raft patches on J-ß2.7 cells expressing I-LFA-1 than on J-ß2.7 cells expressing wt LFA-1. Cells were incubated with TRITC-conjugated Ctx-B, then crosslinked with rabbit anti-Ctx anitbody. Cells were fixed and stained with mAbs against 4ß1, followed by Alexa 488-conjugated goat anti-mouse IgG. (A) Top panels, I-LFA-1-expressing cells; bottom panels, wt LFA-1-expressing cells. (B) Colocalisation of 4ß1 with lipid raft patches. Patches of 4ß1 staining were scored into three categories as described in Fig. 2. The percentages of cells falling into each category are expressed as means±s.d. Data are representative of seven experiments. Black bars, J-ß2.7 cells expressing I-LFA-1; white bars, J-ß2.7 cells expressing wt LFA-1.

View larger version (27K): [in a new window]   Fig. 5. Integrin-mediated adhesion requires intact lipid rafts. (A) Adhesion of Jurkat T cells to immobilised fibronectin using 4ß1 and 5ß1. (B) Adhesion of cultured human T cells to ICAM-1 using LFA-1. Prior to adhesion, the T cells were incubated with no drug (Control), 10 mM MßCD (MbCD), 5 mM MßCD plus 5 mM MßCD-cholesterol (MbCD+Chol), or 10 mM MßCD, followed by washing and incubation with MßCD-cholesterol inclusion complexes (Cholesterol). For the adhesion reaction, cells were incubated with no agonist (Unstim), 100 nM PdBu, 0.5 mM Mn2+ or with the ß2-integrin-activating mAb KIM 185 at 10 µg/ml. Data are representative of four experiments (A) and three experiments (B).

View larger version (50K): [in a new window]   Fig. 6. Depletion of cellular cholesterol by MßCD reduces clustering of 4ß1 on the surface of J-ß2.7 cells expressing I-LFA-1 but has no effect on 4ß1 on wt LFA-1-expressing cells. Cells with (B,D) or without (A,C) pretreatment with 10 mM MßCD were stained on ice with the anti-4 mAb 7.2R, fixed and incubated with Alexa 488-conjugated goat anti-mouse IgG, followed by confocal microscopy. (A,B) J-ß2.7 cells expressing I-LFA-1. (C,D) J-ß2.7 cells expressing wt LFA-1. Single optical sections taken at mid-height of the cells are shown. Data are representative of two experiments. Bar, 10 µm.

View larger version (44K): [in a new window]   Fig. 7. Cytochalasin D treatment affects colocalisation with lipid raft patches of LFA-1, 4ß1 and DAF, but has no effect on the distribution of TfR. Cells were preincubated with 5 µM cytochalasin D, then subjected to lipid raft patching and cell surface protein staining, as in Fig. 1: LFA-1 (A); 4ß1 (B); DAF (C); TfR (D). For each A-D section, top panels, I-LFA-1-expressing cells; bottom panels, wt LFA-1-expressing cells. Cell surface proteins, green; Ctx-B, red. Single optical sections taken at mid-height of the cells are shown. Data are representative of three experiments. Bar, 10 µm.

View larger version (45K): [in a new window]   Fig. 8. Cytochalasin D and latrunculin A treatment affects colocalisation with lipid raft patches of 4ß1 integrin. Cells were preincubated with 5 µM cytochalasin D or 1 µM latrunculin A, then subjected to lipid raft patching and cell surface staining for integrin 4ß1 as in Fig. 7B. (A) 4ß1 integrin on I-LFA-1-expressing cells; (B) 4ß1 integrin on wt LFA-1-expressing cells. Integrin 4ß1, green; Ctx-B, red. Single optical sections taken at mid-height of the cells are shown. Data are representative of three experiments. Bar, 5 µm.