Papel de los microdominios de membrana raft en los procesos de polarización y migración celular

Abstract

Cell migration is an important process for several cell functions. To migrate, cells must acquire and maintain spatial and functional polarity between initially equivalent cell parts. Chemoattractant-stimulated cells develop two opposite poles during the polarization process, the leading edge located at the advance front, and the uropod located at the rear. Several lines of evidence have emphasized the importance of glycosphingolipids and cholesterol-enriched membrane domains –-termed rafts–- in mediating the asymmetric redistribution of specialized molecules in polarized cells. In fact, membrane raft microdomains are proposed as platforms for the selective delivery of membrane proteins to specialized cell surfaces in polarized neurons and epithelial cells. In this report, we show that raft-associated membrane proteins and lipids are distributed asymmetrically in polarized leukocytes and carcinoma cells during chemotaxis. Our data suggest that raft partitioning is a major determinant for asymmetric protein distribution in cells with a migratory phenotype. Ectopically expressed raft-associated proteins are redistributed to the leading edge or to the uropod during cell polarization; conversely, modification of these proteins such that they do not associate with rafts inhibits their asymmetric distribution. We also found that polarized leukocytes segregate leading edge and uropod markers into two different raft types, the L- and U-rafts, which differ in lipid and protein composition, leading to functional specialization of these cell compartments. Lrafts are enriched in GM3 and chemoattractant receptors, whereas U-rafts are enriched in GM1 and cell adhesion receptors. Redistribution of both L- and U-rafts is inhibited by treatment with PTx or actin depolymerizing drugs, suggesting that they require Gi protein signaling and an intact actin cytoskeleton. Moreover, we describe the interaction between the chemoattractant receptor CXCR4 and the actin-binding protein filamin A, which could take place as consequence of the association of these proteins with lipid rafts. It has been suggested that the most important role of rafts at the cell surface is their function in signal transduction. We studied the role of lipid rafts in spatio-temporal signal organization during cell chemotaxis. We used time-lapse confocal microscopy to analyze the dynamic redistribution of molecules in chemoattractant-stimulated leukocytes; this technique confirmed that chemmoattractants induce persistent redistribution of raft domains to both cell poles. The implication of raft reorganization in signaling was studied by analyzing CCR5, PI3K and PTEN redistribution after chemoattractant stimulation. The L-raft-associated chemokine receptor CCR5 redistributes to the leading edge of polarized migrating cells; accumulation of this receptor at the cell front correlates with PI3K p110γ subunit recruitment to L-rafts, where it is subsequently activated. We nonetheless observed that PTEN remains mostly cytoplasmic during cell chemotaxis, with no obvious accumulation in specific cell areas at the resolution level afforded by this technique. Disruption of raft structure by membrane cholesterol sequestration impedes cytoskeleton rearrangement and raft redistribution; concomitantly, this treatment prevents asymmetric recruitment of chemokine receptors and PI3K to the leading edge. The direct consequence is the cellʼs inability to sense a chemoattractant gradient, and consequently to polarize and undergo chemotaxis. These results provide strong evidence that raft domains organize spatial signaling during cell chemotaxis. In summary, we propose that raft domain segregation may be an organizational principle that mediates redistribution of specialized molecules needed for cell chemotaxis.