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Department of Immunology and Oncology, Centro Nacional de Biotecnología (CNB)/CSIC, UAM Campus de Cantoblanco, E-28049 Madrid, Spain

Address correspondence to Santos Mañes, Dept. of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, UAM Campus de Cantoblanco, E-28049 Madrid, Spain. Tel.: 34-91-585-4660. Fax: 34-91-372-0493. email: smanes{at}cnb.uam.es

Spatially restricted activation of signaling molecules governs critical aspects of cell migration; the mechanism by which this is achieved nonetheless remains unknown. Using time-lapse confocal microscopy, we analyzed dynamic redistribution of lipid rafts in chemoattractant-stimulated leukocytes expressing glycosyl phosphatidylinositol–anchored green fluorescent protein (GFP-GPI). Chemoattractants induced persistent GFP-GPI redistribution to the leading edge raft (L raft) and uropod rafts of Jurkat, HL60, and dimethyl sulfoxide–differentiated HL60 cells in a pertussis toxin–sensitive, actin-dependent manner. A transmembrane, nonraft GFP protein was distributed homogeneously in moving cells. A GFP-CCR5 chimera, which partitions in L rafts, accumulated at the leading edge, and CCR5 redistribution coincided with recruitment and activation of phosphatidylinositol-3 kinase in L rafts in polarized, moving cells. Membrane cholesterol depletion impeded raft redistribution and asymmetric recruitment of PI3K to the cell side facing the chemoattractant source. This is the first direct evidence that lipid rafts order spatial signaling in moving mammalian cells, by concentrating the gradient sensing machinery at the leading edge.

Key Words: cell polarization; lipid rafts; chemotaxis; chemokine; phosphatidyl inositol-3 kinase

The online version of this article contains supplemental material.

Abbreviations used in this paper: CD, cyclodextrin; CTx, cholera toxin ß-subunit; cytRFP, cytosolic red fluorescent protein; DRM, detergent-resistant membranes; L raft, leading edge raft; PH, pleckstrin homology; PHAKT-GFP, AKT PH domain fused to GFP; PHAKT-RFP, AKT PH domain fused to DsRed2-FP; PI3K, phosphatidylinositol-3 kinase ; PTx, pertussis toxin; U raft, uropod raft.

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