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First published online July 30, 2004
doi: 10.1242/10.1242/jcs.01270

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Lateral diffusion of Toll-like receptors reveals that they are transiently confined within lipid rafts on the plasma membrane

¹ Infection and Immunity Group, School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, UK
² Department of Biochemical Pharmacology, University of Konstanz, Universitätsstr. 10, 78457 Konstanz, Germany
³ Department of Food Biophysics, Institute of Food Research, Norwich Research Park, Colney Lane, Norwich, NR4 7UA, UK

View larger version (26K): [in a new window]   Fig. 1. TLR2 is present in lipid rafts following LTA stimulation. Monocytes were either not stimulated (A,B) or stimulated with 100 mg/ml LTA in 5% HPS for 30 minutes (C) prior to solubilisation with 1% Triton X-100 buffer for 1 hour on ice and then subjected to sucrose density gradient centrifugation. Fractions were collected from the top of the gradient and equivalent portions of each fraction were analysed by SDS-PAGE and immunoblotting. The lipid raft marker was detected using HRP-conjugated cholera toxin (A), the nitrocellulose membranes were also probed with TLR2 specific mAb (B,C). The relative positions of the raft and non-raft (soluble) fractions are indicated.

View larger version (66K): [in a new window]   Fig. 2. TLR2 and GM-1 ganglioside FRET measurements before and after LTA stimulation. Energy transfer between TLR2 (Cy3-TLR2) and GM-1 ganglioside (Cy5-cholera toxin) before (A,B) and after (C,D) stimulation by LTA can be detected by the increase in donor fluorescence after acceptor photobleaching. (A,C) Donor (Cy3) image after acceptor photobleaching, (B,D) E image (image resulting from the subtraction of the Cy3 image before photobleaching from the Cy3 image after photobleaching). E as a function of fluorescence, for D:A of 1:1 (squares), 1:2 (closed circles) and 1:4 (open circles) (E). Scale bar: 5 µm.

View larger version (16K): [in a new window]   Fig. 3. Disrupting lipid raft integrity inhibits LPS-mediated cellular activation. CHO/CD14/TLR2 reporter cell line was either not stimulated (A) or stimulated with 100 mg/ml of LTA in 5% HPS for 30 min either in the absence (B) or presence (C) or 60 µg/ml nystatin. The induction of CD25 surface expression was detected with FITC-CD25. Fluorescence was detected using a FACSCalibur (BectonDickinson), counting 10,000 cells per sample. (D) The effect of raft-disrupting drugs on TNF- secretion was measured in treated monocytes isolated from the blood of healthy donors with 60 µg/ml nystatin (open circles) prior to stimulation with LTA. Control experiments with cells stimulated with LTA in the absence of raft-disrupting drugs were also performed (closed circles). TNF- secretion was measured using ELISA. Each data point represents the mean±s.d. of three independent experiments.

View larger version (77K): [in a new window]   Fig. 4. Calcium signalling in lipid rafts. CHO cells transfected with CD14 and TLR4 were incubated for 30 minutes in medium supplemented with 2 µM X-Rhod-1 1/AM and 25 mg/l Pluronic F-127. LPS was added at zero time, resulting in the production of Ca2+, which was detected as an increase in X-Rhod 1 fluorescence. Lipid rafts were visualised using Cy5-cholera-toxin (A), TLR4 was visualised using OG-TLR4 (B); Ca2+ signal was visualised by the fluorescence of X-Rhod-1 (C); merged image (D). (E) Ca2+ responses monitored by X-Rhod-1 fluorescence in cells expressing clustered (cell 1, solid line) or not clustered (cell 2, dashed line) TLR4 molecules. (F) Mobility of TLR4 measured by FRAP in cells exhibiting clusters (cell 1, solid line), as well as diffuse (cell 2, dashed line) TLR molecules.

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