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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
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Abstract |
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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
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Introduction |
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Membrane rafts have been characterized as cholesterol- and glycosphingolipid-enriched domains. A role is proposed for rafts in cell migration based on the observation that depletion of plasma membrane cholesterol inhibits cell polarization and migration (Mañes et al., 1999; Khanna et al., 2002). Asymmetric raft domain distribution has also been described after cell stimulation with chemoattractants or with electric fields (Mañes et al., 1999; Gómez-Moutón et al., 2001; Seveau et al., 2001; Millan et al., 2002; Zhao et al., 2002; van Buul et al., 2003). In some reports, this redistribution parallels chemoattractant receptor accumulation at the leading edge (Mañes et al., 1999; Gómez-Moutón et al., 2001; Zhao et al., 2002; van Buul et al., 2003), raising the possibility that rafts act as signal amplification centers during cell polarization and chemotaxis. Uropod raft accumulation is also reported for T cells (Millan et al., 2002) and neutrophils (Seveau et al., 2001). In Jurkat cells, raft subtypes distinguished by ganglioside composition have been identified at each cell pole, with leading edge rafts (L rafts) enriched in GM3, whereas uropod rafts (U rafts) are GM1-enriched (Gómez-Moutón et al., 2001). The evidence for asymmetric raft distribution was obtained in fixed cells; it is consequently not known whether rafts in fact redistribute during directional cell movement.
Here, we used time-lapse confocal microscopy to analyze the dynamic redistribution of raft domains in chemoattractant-stimulated leukocytes. We found that chemoattractants induce persistent redistribution of raft-associated glycosylphosphatidyl inositol (GPI)anchored GFP (GFP-GPI) to both cell edges in a pertussis toxin (PTx)sensitive, actin-dependent manner, confirming L and U raft segregation in polarized leukocytes. The implication of raft reorganization in signaling was studied by analyzing chemoattractant receptor redistribution in chemotaxing cells. We observed that CCR5 redistributed preferentially to the leading edge of polarized migrating cells. This chemoattractant receptor accumulation correlates with phosphatidylinositol-3 kinase (PI3K
) recruitment to L rafts, where it is subsequently activated, as determined by AKT pleckstrin homology (PH) domain recruitment in chemotaxing cells. The results indicate that lipid rafts are platforms for organize spatial signaling during cell chemotaxis, and constitute the first direct evidence of PI3K
polarization in chemotaxing mammalian cells.
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Results |
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Chemoattractant receptors accumulate at the cell front in L rafts
We analyzed whether other raft-associated proteins in addition to GFP-GPI accumulate at the leading edge during chemotaxis. Chemokine receptors are reported to associate with lipid rafts (Mañes et al., 1999, 2000; 2003a; Gómez-Moutón et al., 2001; Sorice et al., 2001; Nguyen and Taub, 2002; Popik et al., 2002; Triantafilou et al., 2002; Viard et al., 2002; Nguyen and Taub, 2003; van Buul et al., 2003; Venkatesan et al., 2003). We constructed GFP-CCR5 chimeras in which the fluorescent protein was tagged to the receptor NH2 or the COOH terminus. Both chimeras partitioned in L rafts, as indicated by exclusive colocalization with GM3 ganglioside (Fig. 4 A), and responded equally to RANTES (CCL5), as indicated by ligand-induced Ca2+ flux (Fig. 4 B). In real-time experiments, we nonetheless found that the COOH terminus GFP-tagged CCR5 chimera internalized in response to ligand and accumulated intracellularly for >60 min (unpublished data), indicating that recycling of this chimera was impaired. This concurs with the observation that the CCR5 COOH terminus is required for appropriate receptor trafficking (Blanpain et al., 2001; Percherancier et al., 2001).
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Chemoattractant-mediated PI3K recruitment and activation take place in L rafts
Because PI3K (class IB) is activated downstream of the chemokine receptors and is required for neutrophil chemotaxis (Stephens et al., 1997; Wymann and Pirola, 1998; Hannigan et al., 2002), we analyzed whether this isoform is distributed asymmetrically in migrating mammalian cells. Confocal videomicroscopy of DMSO-treated HL60 cells coexpressing RFP-GPI and a COOH-terminal GFP-tagged p110
PI3K subunit showed that p110
-GFP is recruited to the cell area facing the fMLP source. Leading edge enrichment in p110
-GFP is closely associated with RFP-GPI redistribution to this site (Fig. 5 A; Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200309101/DC1). Similar experiments performed with cells coexpressing p110
-GFP and the cytosolic red fluorescent protein (cytRFP) showed that persistent p110
accumulation at the leading edge is not solely a consequence of the cytoplasm flow that pushes the cell front (Fig. 5 B; Video 10).
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We next analyzed the dynamics of PHAKT-RFP during chemotaxis as an indirect probe for PI3K activation. PHAKT-RFP is recruited mainly to the leading edge of chemotaxing cells (Fig. 6 A; Video 11, available at http://www.jcb.org/cgi/content/full/jcb.200309101/DC1). Coexpression of AKT PH domain fused to GFP (PHAKT-GFP) with cytRFP suggested that a fraction of the PH domain is closely associated to the plasma membrane (Fig. 6 B). Similar results were obtained for undifferentiated SDF-1stimulated HL60 cells (unpublished data). Treatment with large doses of the PI3K inhibitor LY294002 abolished PHAKT-RFP recruitment to the leading cell edge, as well as its colocalization with GFP-GPI (Fig. 6 C; Video 12). These results suggest that chemoattractants induce PI3K recruitment and activation in lipid rafts.
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Discussion |
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Distinct raft types segregate to the leading edge and uropod in leukocytes
We reported that polarized lymphocytes redistribute GM3-enriched rafts to the leading edge, whereas GM1-based rafts concentrate at the uropod (Gómez-Moutón et al., 2001). This ganglioside segregation is observed in the anterior and posterior parts of HL60 and DMSO-treated HL60 cells (unpublished data). The segregation of distinct raft subtypes to opposite cell poles has been also implicated in pheromone-induced yeast polarization (Bagnat and Simons, 2002), indicating that this complexity is not restricted to leukocytes but probably occurs in many other cell types undergoing polarization. More importantly, we show that persistent L and U raft segregation occurs in live cells engaged in chemotaxis. GFP-GPI colocalizes with GM1 at the uropod and with GM3 at the leading edge (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200309101/DC1), explaining why GFP-GPI labels both cell poles in the time-lapse experiments. Although GFP-GPI has no partitioning bias for a specific raft subtype, there must be molecular signals determining the preferential association of proteins with a specific raft type because the GFP-CCR5 chimera colocalized exclusively with GM3. A possible explanation for this selectivity is that some membrane receptors interact directly with specific lipids, determining their partitioning into specific raft subtypes. For instance, the EGF receptor interacts with GM3 (Miljan et al., 2002) and accumulates at the leading edge during cell electrotaxis (Zhao et al., 2002).
Although our results show GFP-GPI redistribution in all cells studied, relative amounts of this protein vary notably at the front and rear of distinct chemotaxing cell types. Greater GFP-GPI accumulation was observed at the leading edge in Jurkat and in HL60-DMSO cells, whereas undifferentiated HL60 cells showed greater accumulation at the uropod. This variation may reflect the relative size and position of the uropod and the leading edge in these cell types; HL60 cells usually have more prominent uropods than Jurkat or differentiated HL60 cells. These differences may nonetheless represent distinct membrane ganglioside content; HL60 cells have nearly twice as much GM1 as GM3, although this ratio reverses when they are induced to differentiate (Zeng et al., 1995).
Lipid rafts as an organizing platform for signaling during gradient sensing and cell polarization
Current evidence indicates that lipid rafts serve as platforms that increase the efficiency of interactions between activated receptors and signal transduction partners. In migrating cells, lipid rafts may also restrict and/or amplify signaling in specific cell areas. To our knowledge, this paper provides the first direct evidence of the way in which raft domain segregation controls signaling spatially in mammalian cells engaged in chemotaxis. First, we show that raft-associated proteins, including chemoattractant receptors, polarize to specific cell areas in chemotaxing cells. As demonstrated for GFP-GPI and GFP-CCR5, redistribution of raft-associated proteins does not simply reflect plasma membrane accumulation at the leading edge or the uropod due to membrane folding in those areas. Whereas GFP-CCR5 fluorescence concentrated predominantly at the front of polarized cells, the intensity of a membrane probe was similar at the front and the back of the moving cell. This suggests that GFP-CCR5, as well as other L raftassociated proteins, accumulates and persists at the leading edge via an active mechanism that depends on chemoattractant receptor signaling because PTx suppresses raft redistribution in directionally stimulated cells.
Second, we show that accumulation of the L raftassociated GFP-CCR5 receptor at the cell front correlates with recruitment of the PI3K p110 catalytic subunit to the leading edge and an increase in PI3K products at this site. Although both p110
and the AKT PH domain colocalize with the raft probe, we cannot conclude that PI3K activation takes place precisely in L rafts, due to the relatively low resolution of the technique. Nonetheless, we also detect recruitment of the class IB and class IA PI3K to DRM after chemoattractant stimulation, again suggesting that PI3K can be activated in rafts. In contrast to PI3K, we did not detect redistribution of both NH2 and COOH terminus GFP-tagged versions of PTEN (unpublished data).
Finally, we show that cholesterol depletion impedes raft redistribution and, concomitantly, asymmetric PHAKT-RFP recruitment to the cell side facing the chemoattractant source. Chemokine receptor signaling requires association to cholesterol-enriched raft domains (Nguyen and Taub, 2002, 2003). Under the mild cholesterol depletion conditions used here, we observed PH domain recruitment to the membrane, suggesting that G proteinmediated signaling takes place in these cells. The cholesterol-depleted cells can extend small pseudopods, although in random directions; these cells do not recruit PH domains asymmetrically. The results suggest that lipid rafts are involved in cell orientation and polarization toward the attractant source.
We propose that lipid rafts are fundamental elements of the sophisticated guidance system that cells use to orient and move in a chemoattractant gradient. L and U raft segregation permits delivery of "active" receptors to the appropriate cell site, restricting the activation of specific signaling pathways. Our results concur with those of others (Weiner et al., 2002), indicating that signaling molecule relocalization during chemotaxis is the result of interrelated feedback loops. Lipid raft polarization required chemoattractant receptor signaling and actin polymerization, whereas cholesterol depletion prevented asymmetric PI3K activity. Nonetheless, inhibition of PI3K activity also prevented raft redistribution. All these elements would be engaged in positive feedback loops that reinforce the asymmetric sensitivity of the guidance system itself by accumulating chemoattractant receptors at a higher concentration at the cell front. Thus, lipid rafts appear to function as an organizing platform for amplifying intracellular signaling after chemoattractant stimulation.
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Materials and methods |
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Time-lapse confocal videomicroscopy
Real-time cell chemotaxis was studied using time-lapse confocal microscopy. Starved cells were plated for 1 h at 37°C on fibronectin-coated chamber coverslips (Nunc). Cell chemotaxis studies were performed at 37°C using a heating plate and a micromanipulation system (Narishige) adapted to a confocal microscope (Leica). Stimulus was supplied in 12 µm of micropipette prepared in a Kopf pipette puller using thin-wall glass capillaries with an inner filament (Clark Electromedical Instruments), filled with 100 nM SDF-1 (PeproTech), 100 nM RANTES (PeproTech) or 100 nM fMLP (Sigma-Aldrich) in serum-free RPMI 1640 and sealed at the back. Fluorescence and phase contrast images were recorded at established time intervals and resulting videos were processed with NIH-Image J software. Fluorescence scanning was performed with MicroImage software (Olympus Optical Co.).
In some experiments, starved cells were treated with 10 mM of latrunculin-B for 30 min at 37°C (Calbiochem) or 0.5 µg/ml of PTx for 16 h at 37°C (Sigma-Aldrich), washed twice with medium and plated on fibronectin-coated coverslips for chemotaxis. To inhibit PI3K activity, cells were preincubated with 100 µM of LY 294002 for 1 h (Calbiochem) before plating; LY 294002 was maintained at 40 µM during the chemotaxis assay. To deplete cholesterol, serum-starved cells were treated with 12 mM of CD for 30 min at 37°C (Sigma-Aldrich); CD was removed by washing with serum-free medium containing 0.01% BSA, and an aliquot of CD-treated cells was incubated for 30 min at 37°C in RPMI 1640 containing 100 µg/ml of cholesterol (Sigma-Aldrich) and plated on coverslips for chemotaxis. Dark-phase images were taken from eight fields and cells with a polarized morphology were counted.
Immunofluorescence and antibody-induced patching
Jurkat, HL60, and DMSO-treated HL60 cells were plated on fibronectin-coated chambered glass slides (Nunc) 12 h before assay. Cells were starved and stimulated for 10 min at 37°C with 100 nM SDF-1 or 100 nM fMLP, then washed and fixed with 3.7% PFA for 15 min at 20°C in PBS. After fixing, samples were incubated with biotin- or FITC-labeled cholera toxin ß-subunit (CTx) for 5 min at 4°C (Sigma-Aldrich) and an anti-GM3 human polyclonal antiserum for 45 min at 4°C (a gift from E. Gallardo and M. Illa, Santa Creu i Sant Pau Hospital, Barcelona, Spain), followed by Cy2- or Cy3-conjugated second antibodies (Jackson ImmunoResearch) or Cy3-streptavidin. In some experiments, Jurkat cells were stained with the di-unsaturated
9,12-C18 dialkylcarbocyanine (FAST-DiI; Molecular Probes) before stimulation with SDF-1
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For patching experiments, latrunculin-B or PTx-treated cells were incubated for 30 min at 12°C with an anti-GFP antibody (BD Biosciences). Further cross-linking was performed with Alexa 488labeled goat antimouse antibody for 30 min at 12°C. For co-patching, CCR5-GFP or GFP-CCR5expressing Jurkat cells were incubated for 30 min at 12°C with an anti-GFP antibody (CLONTECH Laboratories, Inc.) and biotinylated CTx (Sigma-Aldrich), or with an anti-GM3 antibody. Further cross-linking was performed with the corresponding Cy2- or Cy3-labeled second antibodies. Cells were methanol-fixed for 10 min at -20°C before mounting and confocal analysis.
Calcium determination
Changes in intracellular calcium (Ca2+) concentration were monitored using the fluorescent probe Fluo-3,AM (Molecular Probes). Jurkat cells expressing the NH2- or COOH-terminal GFP-tagged CCR5 chimeras were resuspended in RPMI 1640 containing 10% FBS and incubated with Fluo-3,AM (300 mM in DMSO, 10 µl/106 cells) for 15 min at 37°C. After incubation, cells were washed and resuspended in complete medium containing 2 mM CaCl2 and maintained at 37°C before addition of 10 nM of RANTES. Ca2+ release was determined (37°C, 525 nm) in an EPICS XL flow cytometer (Beckman Coulter).
DRM isolation
DRM were isolated as described previously (Mañes et al., 1999). Normalized protein amounts for each fraction were resolved in SDS-PAGE and analyzed sequentially by blotting with an anti-p85 PI3K (Upstate Biotechnology), an anti-p110 PI3K (a gift from R. Wetzker), anti-GFP, and antitransferrin receptor (Zymed Laboratories) antibodies.
Online supplemental material
Video 1 shows the dynamic redistribution of GFP-GPI during chemotaxis of Jurkat cells. Video 2 shows the dynamic redistribution of GFP-GPI during chemotaxis of HL60 cells. Video 3 shows the dynamic redistribution of GFP-GPI during chemotaxis of HL60-DMSO cells. Video 4 shows the dynamic redistribution of GFP-GT46 during chemotaxis of Jurkat cells. Video 5 shows the dynamic redistribution of GFP-GT46 during chemotaxis of HL60 cells. Video 6 shows the dynamic redistribution of GFP-GPI in PTx-treated Jurkat cells exposed to an SDF-1loaded micropipette. Video 7 shows the dynamic redistribution of GFP-GPI in latrunculin-treated Jurkat cells exposed to an SDF-1
loaded micropipette. Video 8 shows the dynamic redistribution of GFP-CCR5 and PHAKT-RFP chimeras during chemotaxis of Jurkat cells, including single colors and the merge. Video 9 shows the dynamic redistribution of p110
-GFP and RFP-GPI during chemotaxis of HL60-DMSO cells. Single colors and the merge are shown. Video 10 shows the dynamic redistribution of p110
-GFP and cytRFP during chemotaxis of HL60-DMSO cells. Single colors and the merge are shown. Video 11 shows the dynamic redistribution of GFP-GPI and PHAKT-RFP during chemotaxis of HL60-DMSO cells, including single colors and the merge. Video 12 shows the dynamic redistribution of GFP-GPI and PHAKT-RFP during chemotaxis of HL60-DMSO cells pretreated with the PI3K inhibitor LY 294002. Single colors and the merge are shown. Video 13 shows the dynamic redistribution of GFP-GPI and PHAKT-RFP during chemotaxis of HL60-DMSO cells pretreated with methyl-ß-CD. Single colors and the merge are shown. Video 14 shows the dynamic redistribution of GFP-GPI and PHAKT-RFP during chemotaxis of methyl-ß-CDtreated HL60-DMSO cells replenished with cholesterol. Single colors and the merge are shown. Fig. S1 shows GFP-GPI colocalization with GM3 and GM1 in Jurkat cells. Fig. S2 shows single color images for Fig. 4 A. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200309101/DC1.
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Acknowledgments |
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S. Jiménez-Baranda is the recipient of a pre-doctoral fellowship from the Spanish Programa de Formación de Personal Universitario. This work was supported by grants from the Spanish MCyT and European Union (QLG1CT 2001-02171). The Department of Immunology and Oncology was founded and is supported by the Spanish Council for Scientific Research (CSIC) and by Pfizer.
Submitted: 16 September 2003
Accepted: 13 January 2004
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