Altered CXCR4 dynamics at the cell membrane impairs directed cell migration in WHIM syndrome patients.

Chemokine receptor nanoscale organization at the cell membrane is orchestrated by the actin cytoskeleton and influences cell responses. Using single-particle tracking analysis we show that CXCR4R334X, a truncated mutant chemokine receptor linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), fails to nanoclusterize after CXCL12 stimulation, and alters the lateral mobility and spatial organization of CXCR4 when coexpressed. These findings correlate with multiple phalloidin-positive protrusions in cells expressing CXCR4R334X, and their inability to correctly sense chemokine gradients. The underlying mechanisms involve inappropriate actin cytoskeleton remodeling due to the inadequate ß-arrestin1 activation by CXCR4R334X, which disrupts the equilibrium between activated and deactivated cofilin. Overall, we provide insights into the molecular mechanisms governing CXCR4 nanoclustering, signaling and cell function, and highlight the essential scaffold role of ß-arrestin1 to support CXCL12-mediated actin reorganization and receptor clustering. These defects associated with CXCR4R334X expression might contribute to the severe immunological symptoms associated with WHIM syndrome.

Fluctuations in AKT and PTEN Activity Are Linked by the E3 Ubiquitin Ligase cCBL.

3-Poly-phosphoinositides (PIP3) regulate cell survival, division, and migration. Both PI3-kinase (phosphoinositide-3-kinase) and PTEN (phosphatase and tensin-homolog in chromosome 10) control PIP3 levels, but the mechanisms connecting PI3-kinase and PTEN are unknown. Using non-transformed cells, the activation kinetics of PTEN and of the PIP3-effector AKT were examined after the addition of growth factors. Both epidermal growth factor and serum induced the early activation of AKT and the simultaneous inactivation of PTEN (at ~5 min). This PIP3/AKT peak was followed by a general reduction in AKT activity coincident with the recovery of PTEN phosphatase activity (at ~10-15 min). Subsequent AKT peaks and troughs followed. The fluctuation in AKT activity was linked to that of PTEN; PTEN reconstitution in PTEN-null cells restored AKT fluctuations, while PTEN depletion in control cells abrogated them. The analysis of PTEN activity fluctuations after the addition of growth factors showed its inactivation at ~5 min to be simultaneous with its transient ubiquitination, which was regulated by the ubiquitin E3 ligase cCBL (casitas B-lineage lymphoma proto-oncogene). Protein-protein interaction analysis revealed cCBL to be brought into the proximity of PTEN in a PI3-kinase-dependent manner. These results reveal a mechanism for PI3-kinase/PTEN crosstalk and suggest that cCBL could be new target in strategies designed to modulate PTEN activity in cancer.

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy.

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

Remodeling our concept of chemokine receptor function: From monomers to oligomers.

The chemokines direct leukocyte recruitment in both homeostatic and inflammatory conditions, and are therefore critical for immune reactions. By binding to members of the class A G protein-coupled receptors, the chemokines play an essential role in numerous physiological and pathological processes. In the last quarter century, the field has accumulated much information regarding the implications of these molecules in different immune processes, as well as mechanistic insight into the signaling events activated through their binding to their receptors. Here, we will focus on chemokine receptors and how new methodological approaches have underscored the role of their conformations in chemokine functions. Advances in biophysical-based techniques show that chemokines and their receptors act in very complex networks and therefore should not be considered isolated entities. In this regard, the chemokine receptors can form homo- and heterodimers as well as oligomers at the cell surface. These findings are changing our view as to how chemokines influence cell biology, identify partners that regulate chemokine function, and open new avenues for therapeutic intervention.

Separating Actin-Dependent Chemokine Receptor Nanoclustering from Dimerization Indicates a Role for Clustering in CXCR4 Signaling and Function.

A current challenge in cell motility studies is to understand the molecular and physical mechanisms that govern chemokine receptor nanoscale organization at the cell membrane, and their influence on cell response. Using single-particle tracking and super-resolution microscopy, we found that the chemokine receptor CXCR4 forms basal nanoclusters in resting T cells, whose extent, dynamics, and signaling strength are modulated by the orchestrated action of the actin cytoskeleton, the co-receptor CD4, and its ligand CXCL12. We identified three CXCR4 structural residues that are crucial for nanoclustering and generated an oligomerization-defective mutant that dimerized but did not form nanoclusters in response to CXCL12, which severely impaired signaling. Overall, our data provide new insights to the field of chemokine biology by showing that receptor dimerization in the absence of nanoclustering is unable to fully support CXCL12-mediated responses, including signaling and cell function in vivo.

The atypical receptor CCRL2 is required for CXCR2-dependent neutrophil recruitment and tissue damage.

CCRL2 is a 7-transmembrane domain receptor that shares structural and functional similarities with the family of atypical chemokine receptors (ACKRs). CCRL2 is upregulated by inflammatory signals and, unlike other ACKRs, it is not a chemoattractant-scavenging receptor, does not activate ß-arrestins, and is widely expressed by many leukocyte subsets. Therefore, the biological role of CCRL2 in immunity is still unclear. We report that CCRL2-deficient mice have a defect in neutrophil recruitment and are protected in 2 models of inflammatory arthritis. In vitro, CCRL2 was found to constitutively form homodimers and heterodimers with CXCR2, a main neutrophil chemotactic receptor. By heterodimerization, CCRL2 could regulate membrane expression and promote CXCR2 functions, including the activation of ß2-integrins. Therefore, upregulation of CCRL2 observed under inflammatory conditions is functional to finely tune CXCR2-mediated neutrophil recruitment at sites of inflammation.

Use of Lentiviral Particles As a Cell Membrane-Based mFasL Delivery System for In Vivo Treatment of Inflammatory Arthritis.

During budding, lentiviral particles (LVP) incorporate cell membrane proteins in the viral envelope. We explored the possibility of harnessing this process to generate LVP-expressing membrane proteins of therapeutic interest and studied the potential of these tools to treat different pathologies. Fas-mediated apoptosis is central to the maintenance of T cell homeostasis and prevention of autoimmune processes. We prepared LVP that express murine FasL on their surface. Our data indicate that mFasL-bearing LVP induce caspase 3 and 9 processing, cytochrome C release, and significantly more cell death than control LVP in vitro. This cytotoxicity is blocked by the caspase inhibitor Z-VAD. Analysis of the application of these reagents for the treatment of inflammatory arthritis in vivo suggests that FasL-expressing LVP could be useful for therapy in autoimmune diseases such as rheumatoid arthritis, where there is an excess of Fas-expressing activated T cells in the joint. LVP could be a vehicle not only for mFasL but also for other membrane-bound proteins that maintain their native conformation and might mediate biological activities.

Epithelial chemokine CXCL14 synergizes with CXCL12 via allosteric modulation of CXCR4.

The chemokine receptor, CXC chemokine receptor 4 (CXCR4), is selective for CXC chemokine ligand 12 (CXCL12), is broadly expressed in blood and tissue cells, and is essential during embryogenesis and hematopoiesis. CXCL14 is a homeostatic chemokine with unknown receptor selectivity and preferential expression in peripheral tissues. Here, we demonstrate that CXCL14 synergized with CXCL12 in the induction of chemokine responses in primary human lymphoid cells and cell lines that express CXCR4. Combining subactive concentrations of CXCL12 with 100-300 nM CXCL14 resulted in chemotaxis responses that exceeded maximal responses that were obtained with CXCL12 alone. CXCL14 did not activate CXCR4-expressing cells (i.e., failed to trigger chemotaxis and Ca2+ mobilization, as well as signaling via ERK1/2 and the small GTPase Rac1); however, CXCL14 bound to CXCR4 with high affinity, induced redistribution of cell-surface CXCR4, and enhanced HIV-1 infection by >3-fold. We postulate that CXCL14 is a positive allosteric modulator of CXCR4 that enhances the potency of CXCR4 ligands. Our findings provide new insights that will inform the development of novel therapeutics that target CXCR4 in a range of diseases, including cancer, autoimmunity, and HIV.-Collins, P. J., McCully, M. L., Martínez-Muñoz, L., Santiago, C., Wheeldon, J., Caucheteux, S., Thelen, S., Cecchinato, V., Laufer, J. M., Purvanov, V., Monneau, Y. R., Lortat-Jacob, H., Legler, D. F., Uguccioni, M., Thelen, M., Piguet, V., Mellado, M., Moser, B. Epithelial chemokine CXCL14 synergizes with CXCL12 via allosteric modulation of CXCR4.

Use of Resonance Energy Transfer Techniques for In Vivo Detection of Chemokine Receptor Oligomerization.

Since the first reports on chemokine function, much information has been generated on the implications of these molecules in numerous physiological and pathological processes, as well as on the signaling events activated through their binding to receptors. As is the case for other G protein-coupled receptors, chemokine receptors are not isolated entities that are activated following ligand binding; rather, they are found as dimers and/or higher order oligomers at the cell surface, even in the absence of ligands. These complexes form platforms that can be modified by receptor expression and ligand levels, indicating that they are dynamic structures. The analysis of the conformations adopted by these receptors at the membrane and their dynamics is thus crucial for a complete understanding of the function of the chemokines. We focus here on the methodology insights of new techniques, such as those based on resonance energy transfer for the analysis of chemokine receptor conformations in living cells.

Chemokine Detection Using Receptors Immobilized on an SPR Sensor Surface.

Chemokines and their receptors take part in many physiological and pathological processes, and their dysregulated expression is linked to chronic inflammatory and autoimmune diseases, immunodeficiencies, and cancer. The chemokine receptors, members of the G protein-coupled receptor family, are integral membrane proteins, with seven-transmembrane domains that bind the chemokines and transmit signals through GTP-binding proteins. Many assays used to study the structure, conformation, or activation mechanism of these receptors are based on ligand-binding measurement, as are techniques to detect new agonists and antagonists that modulate chemokine function. Such methods require labeling of the chemokine and/or its receptor, which can alter their binding characteristics. Surface plasmon resonance (SPR) is a powerful technique for analysis of the interaction between immobilized receptors and ligands in solution, in real time, and without labeling. SPR measurements nonetheless require expression and purification steps that can alter the conformation, stability, and function of the chemokine and/or the chemokine receptor. In this review, we focus on distinct methods to immobilize chemokine receptors on the surface of an optical biosensor. We expose the advantages and disadvantages of different protocols used and describe in detail the method to retain viral particles as receptor carriers that can be used for SPR determinations.

T Cell Migration in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in joints, associated with synovial hyperplasia and with bone and cartilage destruction. Although the primacy of T cell-related events early in the disease continues to be debated, there is strong evidence that autoantigen recognition by specific T cells is crucial to the pathophysiology of rheumatoid synovitis. In addition, T cells are key components of the immune cell infiltrate detected in the joints of RA patients. Initial analysis of the cytokines released into the synovial membrane showed an imbalance, with a predominance of proinflammatory mediators, indicating a deleterious effect of Th1 T cells. There is nonetheless evidence that Th17 cells also play an important role in RA. T cells migrate from the bloodstream to the synovial tissue via their interactions with the endothelial cells that line synovial postcapillary venules. At this stage, selectins, integrins, and chemokines have a central role in blood cell invasion of synovial tissue, and therefore in the intensity of the inflammatory response. In this review, we will focus on the mechanisms involved in T cell attraction to the joint, the proteins involved in their extravasation from blood vessels, and the signaling pathways activated. Knowledge of these processes will lead to a better understanding of the mechanism by which the systemic immune response causes local joint disorders and will help to provide a molecular basis for therapeutic strategies.

Type I phosphatidylinositol 4-phosphate 5-kinase homo- and heterodimerization determines its membrane localization and activity.

Type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KIs; α, ß, and γ) are a family of isoenzymes that produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] using phosphatidylinositol 4-phosphate as substrate. Their structural homology with the class II lipid kinases [type II phosphatidylinositol 5-phosphate 4-kinase (PIP4KII)] suggests that PIP5KI dimerizes, although this has not been formally demonstrated. Neither the hypothetical structural dimerization determinants nor the functional consequences of dimerization have been studied. Here, we used Förster resonance energy transfer, coprecipitation, and ELISA to show that PIP5KIß forms homo- and heterodimers with PIP5KIγ_i2 in vitro and in live human cells. Dimerization appears to be a general phenomenon for PIP5KI isoenzymes because PIP5KIß/PIP5KIα heterodimers were also detected by mass spectrometry. Dimerization was independent of actin cytoskeleton remodeling and was also observed using purified proteins. Mutagenesis studies of PIP5KIß located the dimerization motif at the N terminus, in a region homologous to that implicated in PIP4KII dimerization. PIP5KIß mutants whose dimerization was impaired showed a severe decrease in PI(4,5)P2 production and plasma membrane delocalization, although their association to lipid monolayers was unaltered. Our results identify dimerization as an integral feature of PIP5K proteins and a central determinant of their enzyme activity.

Methods to immobilize GPCR on the surface of SPR sensors.

The G protein-coupled receptors (GPCRs) form one of the largest membrane receptor families. The nature of the ligands that interact with these receptors is highly diverse; they include light, peptides and hormones, neurotransmitters, and small molecular weight compounds. The GPCRs are involved in a wide variety of physiological processes and thus hold considerable therapeutic potential.GPCR function is usually determined in cell-based assays, whose complexity nonetheless limits their use. The use of alternative, cell-free assays is hampered by the difficulties in purifying these seven-transmembrane domain receptors without altering their functional properties. Several methods have been proposed to immobilize GPCR on biosensor surfaces which use antibodies or avidin-/biotin-based capture procedures, alone or with reconstitution of the GPCR physiological microenvironment. Here we propose a method for GPCR immobilization in their native membrane microenvironment that requires no manipulation of the target receptor and maintains the many conformations GPCR can adopt in the cell membrane.

CCR5/CD4/CXCR4 oligomerization prevents HIV-1 gp120IIIB binding to the cell surface.

CCR5 and CXCR4, the respective cell surface coreceptors of R5 and X4 HIV-1 strains, both form heterodimers with CD4, the principal HIV-1 receptor. Using several resonance energy transfer techniques, we determined that CD4, CXCR4, and CCR5 formed heterotrimers, and that CCR5 coexpression altered the conformation of both CXCR4/CXCR4 homodimers and CD4/CXCR4 heterodimers. As a result, binding of the HIV-1 envelope protein gp120IIIB to the CD4/CXCR4/CCR5 heterooligomer was negligible, and the gp120-induced cytoskeletal rearrangements necessary for HIV-1 entry were prevented. CCR5 reduced HIV-1 envelope-induced CD4/CXCR4-mediated cell-cell fusion. In nucleofected Jurkat CD4 cells and primary human CD4(+) T cells, CCR5 expression led to a reduction in X4 HIV-1 infectivity. These findings can help to understand why X4 HIV-1 strains infection affect T-cell types differently during AIDS development and indicate that receptor oligomerization might be a target for previously unidentified therapeutic approaches for AIDS intervention.

CXCL12-mediated murine neural progenitor cell movement requires PI3Kß activation.

The migratory route of neural progenitor/precursor cells (NPC) has a central role in central nervous system development. Although the role of the chemokine CXCL12 in NPC migration has been described, the intracellular signaling cascade involved remains largely unclear. Here we studied the molecular mechanisms that promote murine NPC migration in response to CXCL12, in vitro and ex vivo. Migration was highly dependent on signaling by the CXCL12 receptor, CXCR4. Although the JAK/STAT pathway was activated following CXCL12 stimulation of NPC, JAK activity was not necessary for NPC migration in vitro. Whereas CXCL12 activated the PI3K catalytic subunits p110α and p110ß in NPC, only p110ß participated in CXCL12-mediated NPC migration. Ex vivo experiments using organotypic slice cultures showed that p110ß blockade impaired NPC exit from the medial ganglionic eminence. In vivo experiments using in utero electroporation nonetheless showed that p110ß is dispensable for radial migration of pyramidal neurons. We conclude that PI3K p110ß is activated in NPC in response to CXCL12, and its activity is necessary for immature interneuron migration to the cerebral cortex.

Suppressor of cytokine signaling 1 blocks mitosis in human melanoma cells.

Hypermethylation of SOCS genes is associated with many human cancers, suggesting a role as tumor suppressors. As adaptor molecules for ubiquitin ligases, SOCS proteins modulate turnover of numerous target proteins. Few SOCS targets identified so far have a direct role in cell cycle progression; the mechanism by which SOCS regulate the cell cycle thus remains largely unknown. Here we show that SOCS1 overexpression inhibits in vitro and in vivo expansion of human melanoma cells, and that SOCS1 associates specifically with Cdh1, triggering its degradation by the proteasome. Cells therefore show a G1/S transition defect, as well as a secondary blockade in mitosis and accumulation of cells in metaphase. SOCS1 expression correlated with a reduction in cyclin D/E levels and an increase in the tumor suppressor p19, as well as the CDK inhibitor p53, explaining the G1/S transition defect. As a result of Cdh1 degradation, SOCS1-expressing cells accumulated cyclin B1 and securin, as well as apparently inactive Cdc20, in mitosis. Levels of the late mitotic Cdh1 substrate Aurora A did not change. These observations comprise a hitherto unreported mechanism of SOCS1 tumor suppression, suggesting this molecule as a candidate for the design of new therapeutic strategies for human melanoma.

EBI2 regulates CXCL13-mediated responses by heterodimerization with CXCR5.

B-cell movement into lymphoid follicles depends on the expression of the chemokine receptor CXCR5 and the recently reported Epstein-Barr virus-induced receptor 2 (EBI2). In cooperation with CXCR5, EBI2 helps to position activated B cells in the follicle, although the mechanism is poorly understood. Using human HEK293T cells and fluorescence resonance energy transfer (FRET) techniques, we demonstrate that CXCR5 and EBI2 form homo- and heterodimers. EBI2 expression modulated CXCR5 homodimeric complexes, as indicated by the FRET(50) value (CXCR5 homodimer, 0.9851±0.0784; CXCR5 homodimer+EBI2, 1.7320±0.4905; P<0.05). HEK293T cells expressing CXCR5/EBI2 and primary activated murine B cells both down-modulated CXCR5-mediated responses, such as Ca(2+) flux, cell migration, and MAPK activation; this modulation did not occur when primary B cells were obtained from EBI2(-/-) mice. The mechanism involves a reduction in binding affinity of the ligand (CXCL13) for CXCR5 (K(D): 5.05×10(-8) M for CXCR5 alone vs. 1.49×10(-7) M for CXCR5/EBI2) and in the efficacy (E(max)) of G-protein activation in CXCR5/EBI2-coexpressing cells (42.33±4.3%; P<0.05). These findings identify CXCR5/EBI2 heterodimers as functional units that contribute to the plasticity of CXCL13-mediated B-cell responses.

CD69 modulates sphingosine-1-phosphate-induced migration of skin dendritic cells.

In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69-/- DCs, which presented an increased migration to peripheral LNs. Two-photon microscopy analysis showed that once DCs reached the LNs, CD69 deficiency did not alter DC interstitial motility in the LNs. Chemotaxis to sphingosine-1-phosphate (S1P) was enhanced in CD69-/- DCs compared with wild-type DCs. Accordingly, we detected a higher expression of S1P receptor type-1 (S1P(1)) by CD69-/- DCs, whereas S1P(3) expression levels were similar in wild-type and CD69-/- DCs. Moreover, in vivo treatment with S1P analogs SEW2871 and FTY720 during skin sensitization reduced skin DC migration to peripheral LNs. These results suggest that CD69 regulates S1P-induced skin DC migration by modulating S1P(1) function. Together, our findings increase our knowledge on DC trafficking patterns in the skin, enabling the development of new directed therapies using DCs for antigen (Ag) delivery.

Dynamic regulation of CXCR1 and CXCR2 homo- and heterodimers.

Although homo- and heterodimerization are reported for some chemokine receptors, it remains unclear whether these functional states are in dynamic equilibrium and how receptor/ligand levels influence oligomerization. In human neutrophils and in cell lines that coexpress the chemokine receptors CXCR1 and CXCR2, we used fluorescence resonance energy transfer techniques to show that these two receptors form homo- and heterodimers. Receptor expression and ligand activation were found to regulate the balance between these complexes, adapting the response to changes in the milieu. CXCL8, a ligand for both receptors, alters heterodimeric complexes, whereas it stabilizes homodimers and promotes receptor internalization. Oligomerization of receptors, together with the regulation of their expression and desensitization, could thus contribute to the fine control of chemokine functions.