Understanding Streaming in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>: Theory Versus Experiments

Recent experimental work involving Dictyostelium discoideum seems to contradict several theoretical models. Experiments suggest that localization of the release of the chemoattractant cyclic adenosine monophosphate to the uropod of the cell is important for stream formation during aggregation. Yet several mathematical models are able to reproduce streaming as the cells aggregate without taking into account localization of the chemoattractant. A careful analysis of the experiments and the theory suggests the two major features of the system which are important to stream formation are random cell motion and chemotaxis to regions of higher cell density. Random cell motion acts to reduce streaming, whereas chemotaxis to regions of higher cell density reinforces streaming. With this understanding, the experimental results can be explained in a manner consistent with the theoretical results. In all the experiments, alterations in the two main factors of random motion and chemotaxis to regions of higher cell density, not the localization of the release of the chemoattractant, can explain the results as they relate to streaming. Additionally, a comparison of results from a mathematical model that simulates cells which localize the chemoattractant and cells which do not shows little difference in the streaming patterns.     

Flotillin microdomains interact with the cortical cytoskeleton to control uropod formation and neutrophil recruitment

We studied the function of plasma membrane microdomains defined by the proteins flotillin 1 and flotillin 2 in uropod formation and neutrophil chemotaxis. Flotillins become concentrated in the uropod of neutrophils after exposure to chemoattractants such as N-formyl-Met-Leu-Phe (fMLP). Here, we show that mice lacking flotillin 1 do not have flotillin microdomains, and that recruitment of neutrophils toward fMLP in vivo is reduced in these mice. Ex vivo, migration of neutrophils through a resistive matrix is reduced in the absence of flotillin microdomains, but the machinery required for sensing chemoattractant functions normally. Flotillin microdomains specifically associate with myosin IIa, and spectrins. Both uropod formation and myosin IIa activity are compromised in flotillin 1 knockout neutrophils. We conclude that the association between flotillin microdomains and cortical cytoskeleton has important functions during neutrophil migration, in uropod formation, and in the regulation of myosin IIa.     

Flotillins Interact with PSGL-1 in Neutrophils and,upon Stimulation,Rapidly Organize into Membrane Domains Subsequently Accumulating in the Uropod

Background

Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that are ubiquitously enriched in detergent resistant membranes and are thought to shape membrane microdomains by forming homo- and hetero-oligomers. It was the goal of this study to investigate dynamic membrane microdomain reorganization during neutrophil activation.

Methodology/Principal Findings

We show now, using immunofluorescence staining and co-immunoprecipitation, that endogenous flotillin-1 and -2 colocalize and associate in resting spherical and polarized primary neutrophils. Flotillins redistribute very early after chemoattractant stimulation, and form distinct caps in more than 90% of the neutrophils. At later time points flotillins accumulate in the uropod of polarized cells. Chemotactic peptide-induced redistribution and capping of flotillins requires integrity and dynamics of the actin cytoskeleton, but does not involve Rho-kinase dependent signaling related to formation of the uropod. Both flotillin isoforms are involved in the formation of this membrane domain, as uropod location of exogenously expressed flotillins is dramatically enhanced by co-overexpression of tagged flotillin-1 and -2 in differentiated HL-60 cells as compared to cells expressing only one tagged isoform.Flotillin-1 and -2 associate with P-selectin glycoprotein ligand 1 (PSGL-1) in resting and in stimulated neutrophils as shown by colocalization and co-immunoprecipitation. Neutrophils isolated from PSGL-1-deficient mice exhibit flotillin caps to the same extent as cells isolated from wild type animals, implying that PSGL-1 is not required for the formation of the flotillin caps. Finally we show that stimulus-dependent redistribution of other uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1.

Conclusions/Significance

These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil uropod formation and/or stabilization and organize uropod localization of PSGL-1.     

Localization of chemotactic peptide receptors on rabbit neutrophils

The chemotaxis of blood leukocytes is initiated by the binding of a chemoattractant to specific receptors on the leukocyte cell surface. Although a great deal is known about the biochemical and morphological events accompanying chemotactic activation, there is very little morphological information about the chemoattractant receptors themselves. This latter information is needed so that we may understand the mechanism by which these inflammatory cells detect and respond to chemical gradients. One class of chemotactic factors extensively used to characterize the complex behavioral responses following leukocyte activation are the synthetic formylmethionyl peptides. These peptides, now known to be the analogs of the naturally occurring N-terminal peptides produced by bacteria, are released into culture medium and are believed to be responsible, at least in part, for the accumulation of leukocytes at the sites of bacterial infection. We have localized the receptors for the chemotactic hexapeptide N-formylnorleucyl-leucyl-phenylalanine-norleucyl-[125I]tyrosyl-lys ine [N-fNle-Leu-Phe-Nle-[125I]Tyr-Lys] on whole rabbit peritoneal neutrophils (PMN) using light microscope autoradiography. By this method, the inherent formylpeptide receptor distribution on cells incubated at 4 degrees C appears to be uniform over the surface of both rounded and structurally polarized PMN. Following a short 37 degrees C incubation, cells retain a large proportion of labelled hexapeptide at or near the cell surface and, in addition, polarized PMN redistribute the hexapeptide anteriorly away from the cell uropod.     

Two complementary, local excitation, global inhibition mechanisms acting in parallel can explain the chemoattractant-induced regulation of PI(3,4,5)P3 response in dictyostelium cells

Chemotaxing cells, such as Dictyostelium and mammalian neutrophils, sense shallow chemoattractant gradients and respond with highly polarized changes in cell morphology and motility. Uniform chemoattractant stimulation induces the transient translocations of several downstream signaling components, including phosphoinositide 3-kinase (PI3K), tensin homology protein (PTEN), and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). In contrast, static spatial chemoattractant gradients elicit the persistent, amplified localization of these molecules. We have proposed a model in which the response to chemoattractant is regulated by a balance of a local excitation and a global inhibition, both of which are controlled by receptor occupancy. This model can account for both the transient and spatial responses to chemoattractants, but alone does not amplify the external gradient. In this article, we develop a model in which parallel local excitation, global inhibition mechanisms control the membrane binding of PI3K and PTEN. Together, the action of these enzymes induces an amplified PI(3,4,5)P3 response that agrees quantitatively with experimentally obtained plekstrin homology-green fluorescent protein distributions in latrunculin-treated cells. We compare the model's performance with that of several mutants in which one or both of the enzymes are disrupted. The model accounts for the observed response to multiple, simultaneous chemoattractant cues and can recreate the cellular response to combinations of temporal and spatial stimuli. Finally, we use the model to predict the response of a cell where only a fraction is stimulated by a saturating dose of chemoattractant.     

Microtubule asymmetry during neutrophil polarization and migration

The development of cell polarity in response to chemoattractant stimulation in human polymorphonuclear neutrophils (PMNs) is characterized by the rapid conversion from round to polarized morphology with a leading lamellipod at the front and a uropod at the rear. During PMN polarization, the microtubule (MT) array undergoes a dramatic reorientation toward the uropod that is maintained during motility and does not require large-scale MT disassembly or cell adhesion to the substratum. MTs are excluded from the leading lamella during polarization and motility, but treatment with a myosin light chain kinase inhibitor (ML-7) or the actin-disrupting drug cytochalasin D causes an expansion of the MT array and penetration of MTs into the lamellipod. Depolymerization of the MT array before stimulation caused 10% of the cells to lose their polarity by extending two opposing lateral lamellipodia. These multipolar cells showed altered localization of a leading lamella-specific marker, talin, and a uropod-specific marker, CD44. In summary, these results indicate that F-actin- and myosin II-dependent forces lead to the development and maintenance of MT asymmetry that may act to reinforce cell polarity during PMN migration.     

Chemokines regulate cellular polarization and adhesion receptor redistribution during lymphocyte interaction with endothelium and extracellular matrix. Involvement of cAMP signaling pathway

Leukocyte recruitment is a key step in the inflammatory reaction. Several changes in the cell morphology take place during lymphocyte activation and migration: spheric-shaped resting T cells become polarized during activation, developing a well defined cytoplasmic projection designated as cellular uropod. We found that the chemotactic and proinflammatory chemokines RANTES, MCP-1, and, to a lower extent, MIP-1 alpha, MIP-1 beta, and IL-8, were able to induce uropod formation and ICAM-3 redistribution in T lymphoblasts adhered to ICAM-1 or VCAM- 1. A similar chemokine-mediated effect was observed during T cells binding to the fibronectin fragments of 38- and 80-kD, that contain the binding sites for the integrins VLA-4 and VLA-5, respectively. The uropod structure concentrated the ICAM-3 adhesion molecule (a ligand for LFA-1), and emerged to the outer milieu from the area of contact between lymphocyte and protein ligands. In addition, we found that other adhesion molecules such as ICAM-1, CD43, and CD44, also redistributed to the lymphocyte uropod upon RANTES stimulation, whereas a wide number of other cell surface receptors did not redistribute. Chemokines displayed a selective effect among different T cell subsets; MIP-1 beta had more potent action on CD8+ T cells and tumor infiltrating lymphocytes (TIL), whereas RANTES and MIP-1 alpha targeted selectively CD4+ T cells. We have also examined the involvement of cAMP signaling pathway in uropod formation. Interestingly, several cAMP agonists were able to induce uropod formation and ICAM-3 redistribution, whereas H-89, a specific inhibitor of the cAMP- dependent protein kinase, abrogated the chemokine-mediated uropod formation, thus pointing out a role for cAMP-dependent signaling in the development of this cytoplasmic projection. Since the lymphocyte uropod induced by chemokines was completely abrogated by Bordetella pertussis toxin, the formation of this membrane projection appears to be dependent on G proteins signaling pathways. In addition, the involvement of myosin-based cytoskeleton in uropod formation and ICAM-3 redistribution in response to chemokines was suggested by the prevention of this phenomenon with the myosin-disrupting agent butanedione monoxime. Interestingly, this agent also inhibited the ICAM- 3-mediated cell aggregation, but not the cell adhesion to substrata. Altogether, these results demonstrate that uropod formation and adhesion receptor redistribution is a novel function mediated by chemokines; this phenomenon may represent a mechanism that significantly contributes to the recruitment of circulating leukocytes to inflammatory foci.     

Type Igamma PIP kinase is a novel uropod component that regulates rear retraction during neutrophil chemotaxis

Cell polarization is necessary for directed migration and leukocyte recruitment to inflamed tissues. Recent progress has been made in defining the molecular mechanisms that regulate chemoattractant-induced cell polarity during chemotaxis, including the contribution of phosphoinositide 3-kinase (PI3K)-dependent phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] synthesis at the leading edge. However, less is known about the molecular composition of the cell rear and how the uropod functions during cell motility. Here, we demonstrate that phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma661), which generates PtdIns(4,5)P(2), is enriched in the uropod during chemotaxis of primary neutrophils and differentiated HL-60 cells (dHL-60). Using time-lapse microscopy, we show that enrichment of PIPKIgamma661 at the cell rear occurs early upon chemoattractant stimulation and is persistent during chemotaxis. Accordingly, we were able to detect enrichment of PtdIns(4,5)P(2) at the uropod during chemotaxis. Overexpression of kinase-dead PIPKIgamma661 compromised uropod formation and rear retraction similar to inhibition of ROCK signaling, suggesting that PtdIns(4,5)P(2) synthesis is important to elicit the backness response during chemotaxis. Together, our findings identify a previously unknown function for PIPKIgamma661 as a novel component of the backness signal that regulates rear retraction during chemotaxis.     

Phosphoinositide 3-kinase activity controls the chemoattractant-mediated activation and adaptation of adenylyl cyclase

The binding of chemoattractants to cognate G protein-coupled receptors activates a variety of signaling cascades that provide spatial and temporal cues required for chemotaxis. When subjected to uniform stimulation, these responses are transient, showing an initial peak of activation followed by a period of adaptation, in which activity subsides even in the presence of stimulus. A tightly regulated balance between receptor-mediated stimulatory and inhibitory pathways controls the kinetics of activation and subsequent adaptation. In Dictyostelium, the adenylyl cyclase expressed during aggregation (ACA), which synthesizes the chemoattractant cAMP, is essential to relay the signal to neighboring cells. Here, we report that cells lacking phosphoinositide 3-kinase (PI3K) activity are deficient in signal relay. In LY294002-treated cells, this defect is because of a loss of ACA activation. In contrast, in cells lacking PI3K1 and PI3K2, the signal relay defect is because of a loss of ACA adaptation. We propose that the residual low level of 3-phosphoinositides in pi3k(1-/2-) cells is sufficient to generate the initial peak of ACA activity, yet is insufficient to sustain the inhibitory phase required for its adaptation. Thus, PI3K activity is poised to regulate both ACA activation and adaptation, thereby providing a link to ensure the proper balance of counteracting signals required to maintain optimal chemoresponsiveness.     

The Two Poles of the Lymphocyte: Specialized Cell Compartments for Migration and Recruitment

Chemotaxis, the directed migration of leukocytes towards a chemoattractant gradient, is a key phenomenon in the immune response. During lymphocyte-endothelial and – extracellular matrix interactions, chemokines induce the polarization of T lymphocytes. with generation of specialized cell compartments. The chemokine receptors involved in detection of the chemoattractant gradients concentrate at the leading edge (advancing front or anterior pole) of the cell. The adhesion molecules ICAM- 1, -3, CD44 and CD43 redistribute to the uropod, an appendage at the posterior pole of migrating T lymphocyte that protrudes from the contact area with endothelial or extracellular matrix substrates. Whereas chemokine receptors sense the direction of migration, the uropod is involved in the recruitment of bystander leukocytes through LFA-1/ICAM-dependent cell cell interactions. While β-actin concentrates preferentially at the cell's leading edge, the motor protein myosin II and a microtubule organizing center (MTOC) are packed in the uropod. The actin-binding protein moesin, which belongs to the ERM family of ezrin, radixin and moesin, redistributes to the distal portion of uropods and physically interacts with ICAM-3, CD44 and CD43, thus acting as a physical link between the membrane molecules and the actin cytoskeleton. Moreover, the moesin-ICAM-3 association correlates with the degree of cell polarity. The redistribution of the chemokine receptors and adhesion molecules to opposite poles of the cell in response to a chemoattractant gradient may guide cell migration and cell-cell interactions during lymphoid cell trafficking in immune and inflammatory responses.     

A discrete cell model with adaptive signalling for aggregation of Dictyostelium discoideum.

Dictyostelium discoideum (Dd) is a widely studied model system from which fundamental insights into cell movement, chemotaxis, aggregation and pattern formation can be gained. In this system aggregation results from the chemotactic response by dispersed amoebae to a travelling wave of the chemoattractant cAMP. We have developed a model in which the cells are treated as discrete points in a continuum field of the chemoattractant, and transduction of the extracellular cAMP signal into the intracellular signal is based on the G protein model developed by Tang & Othmer. The model reproduces a number of experimental observations and gives further insight into the aggregation process. We investigate different rules for cell movement the factors that influence stream formation the effect on aggregation of noise in the choice of the direction of movement and when spiral waves of chemoattractant and cell density are likely to occur. Our results give new insight into the origin of spiral waves and suggest that streaming is due to a finite amplitude instability.     

Biogenesis of the posterior pole is mediated by the exosome/microvesicle protein-sorting pathway

Biogenesis of the posterior pole is critical to directed cell migration and other polarity-dependent processes. We show here that proteins are targeted to the posterior pole on the basis of higher order oligomerization and plasma membrane binding, the same elements that target proteins to exosomes/microvesicles (EMVs), HIV, and other retrovirus particles. We also demonstrate that the polarization of the EMV protein-sorting pathway can occur in morphologically non-polarized cells, defines the site of uropod formation, is induced by increased expression of EMV cargo proteins, and is evolutionarily conserved between humans and the protozoan Dictyostelium discoideum. Based on these results, we propose a mechanism of posterior pole biogenesis in which elevated levels of EMV cargoes (i) polarize the EMV protein-sorting pathway, (ii) generate a nascent posterior pole, and (iii) prime cells for signal-induced biogenesis of a uropod. This model also offers a mechanistic explanation for the polarized budding of EMVs and retroviruses, including HIV.     

Cytoskeletal rearrangement during migration and activation of T lymphocytes.

T lymphocytes have an inherent ability to migrate along a chemotactic gradient, which enables them to exit the bloodstream and reach different tissues. Motile T cells display a polarized morphology with two distinct cell compartments: the leading edge and the uropod. During cell polarization, chemoattractant receptors, cell-adhesion molecules and cytoskeletal proteins are redistributed within these cellular compartments. The polarity of T lymphocytes changes during the establishment of antigen-specific cell-cell interactions, and this involves rearrangement of cytoskeletal proteins. This article discusses the regulation of these cytoskeletal rearrangements, and their role in the activation, migration and effector function of T cells.     

Nonuniform distribution of concanavalin-A receptors and surface antigens on uropod-forming thymocytes

Uropods can form spontaneously in a variable fraction of mouse thymocytes incubated for 30--60 min in vitro at temperatures between about 8 degrees and 37 degrees C. The majority of the cells with a typical uropod are medium and large thymocytes. The "normal" distribution of concanavalin-A receptors and antigens recognized by a rabbit anti-mouse thymocyte serum was studied on these cells by electron microscopy using ferritin-conjugated lectin or antibodies. The cells were fixed with glutaraldehyde or formaldehyde before labeling. The distribution was essentially uniform on spherical cells. On the contrary, on cells which had formed a uropod the labeled receptors and antigens appeared to be preferentially concentrated around the nucleus, and depleted over the uropod, and especially over the constriction at the base of the uropod. Uropod formation and inhomogeneous distribution were inhibited or reversed by cytochalasin B, but not by vinblastine or colchicine. When the same ligands were applied to unfixed cells, the labeled and cross-linked components capped normally towards the cytoplasmic pole of the cell. These observations are described in relation to the ability of receptors and antigens to interact with an intracellular mechanical structure, and to the mechanism of capping.     

Chemotactically-induced redistribution of CD43 as related to polarity and locomotion of human polymorphonuclear leucocytes

Leukocyte motility involves pseudopods extension at the leading edge and uropod contraction at the cell rear. Previous studies have shown that the glycoprotein CD43 redistributes to the uropod, when the cells develop polarity and locomotion. The present study addresses the question whether the accumulation of specific membrane molecules, such as CD43 at the contracted uropod precedes or follows development of polarity and locomotion. PMNs were labeled with fluorescent anti-CD43 antibodies and guided to polarize in the direction of a chemoattractant-containing micropipette or, once polarized, they were forced to reverse polarity and movement direction by placing the micropipette behind the uropod. This chemotactically-induced reversal of polarity was used as an efficient tool to analyse the sequence of events. CD43, but not another abundant surface glycoprotein CD45, was concentrated at the uropod. This documents that CD43 redistribution is a selective phenomenon. During reversal of polarity and of locomotion direction, the geometric center of the cell clearly changed direction earlier than the center of anti-CD43 fluorescence intensity. Thus, CD43 redistribution to the new uropod follows rather than precedes reversal of polarity, suggesting that CD43 redistribution is a consequence rather than a prerequisite for polarity and locomotion. PMNs making a U-turn maintained the pre-existing polarity and CD43 remained concentrated at the uropod, even when the front was moving in the opposite direction. Our data show that anterior pseudopod formation, rather than capping of CD43 at the uropod or the position of the uropod determines the direction of locomotion.     

Actin cross-linking proteins cortexillin I and II are required for cAMP signaling during Dictyostelium chemotaxis and development

Starvation induces Dictyostelium amoebae to secrete cAMP, toward which other amoebae stream, forming multicellular mounds that differentiate and develop into fruiting bodies containing spores. We find that the double deletion of cortexillin (ctx) I and II alters the actin cytoskeleton and substantially inhibits all molecular responses to extracellular cAMP. Synthesis of cAMP receptor and adenylyl cyclase A (ACA) is inhibited, and activation of ACA, RasC, and RasG, phosphorylation of extracellular signal regulated kinase 2, activation of TORC2, and stimulation of actin polymerization and myosin assembly are greatly reduced. As a consequence, cell streaming and development are completely blocked. Expression of ACA-yellow fluorescent protein in the ctxI/ctxII-null cells significantly rescues the wild-type phenotype, indicating that the primary chemotaxis and development defect is the inhibition of ACA synthesis and cAMP production. These results demonstrate the critical importance of a properly organized actin cytoskeleton for cAMP-signaling pathways, chemotaxis, and development in Dictyostelium.     

The PI3K-mediated activation of CRAC independently regulates adenylyl cyclase activation and chemotaxis

The ability of a cell to detect an external chemical signal and initiate a program of directed migration along a gradient comprises the fundamental process called chemotaxis. Investigations in Dictyostelium discoideum and neutrophils have established that pleckstrin homology (PH) domain-containing proteins that bind to the PI3K products PI(3,4)P2 and PI(3,4,5)P3, such as CRAC (cytosolic regulator of adenylyl cyclase) and Akt/PKB, translocate specifically to the leading edge of chemotaxing cells. CRAC is essential for the chemoattractant-mediated activation of the adenylyl cyclase ACA, which converts ATP into cAMP, the primary chemoattractant for D. discoideum. The mechanisms by which CRAC activates ACA remain to be determined. We now show that in addition to its essential role in the activation of ACA, CRAC is involved in regulating chemotaxis. Through mutagenesis, we show that these two functions are independently regulated downstream of PI3K. A CRAC mutant that has lost the capacity to bind PI3K products does not support chemotaxis and shows minimal ACA activation. Finally, overexpression of CRAC and various CRAC mutants show strong effects on ACA activation with little effect on chemotaxis. These findings establish that chemoattractant-mediated activation of PI3K is important for the CRAC-dependent regulation of both chemotaxis and adenylyl cyclase activation.     

ICAMs Redistributed by Chemokines to Cellular Uropods as a Mechanism for Recruitment of T Lymphocytes

The recruitment of leukocytes from the bloodstream is a key step in the inflammatory reaction, and chemokines are among the main regulators of this process. During lymphocyte–endothelial interaction, chemokines induce the polarization of T lymphocytes, with the formation of a cytoplasmic projection (uropod) and redistribution of several adhesion molecules (ICAM-1,-3, CD43, CD44) to this structure. Although it has been reported that these cytokines regulate the adhesive state of integrins in leukocytes, their precise mechanisms of chemoattraction remain to be elucidated. We have herein studied the functional role of the lymphocyte uropod. Confocal microscopy studies clearly showed that cell uropods project away from the cell bodies of adhered lymphocytes and that polarized T cells contact other T cells through the uropod structure. Time-lapse videomicroscopy studies revealed that uropod-bearing T cells were able, through this cellular projection, to contact, capture, and transport additional bystander T cells. Quantitative analysis revealed that the induction of uropods results in a 5–10-fold increase in cell recruitment. Uropod-mediated cell recruitment seems to have physiological relevance, since it was promoted by both CD45R0⁺ peripheral blood memory T cells as well as by in vivo activated lymphocytes. Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process. To determine whether the increment in cell recruitment mediated by uropod may affect the transendothelial migration of T cells, we carried out chemotaxis assays through confluent monolayers of endothelial cells specialized in lymphocyte extravasation. An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti– ICAM-3 mAbs or drugs that impair uropod formation. These data indicate that the cell interactions mediated by cell uropods represent a cooperative mechanism in lymphocyte recruitment, which may act as an amplification system in the inflammatory response.     

Dynamic redistribution of raft domains as an organizing platform for signaling during cell chemotaxis

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 gamma 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.