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.     

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.     

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.     

A novel serine-rich motif in the intercellular adhesion molecule 3 is critical for its ezrin/radixin/moesin-directed subcellular targeting.

Intercellular adhesion molecule 3 (ICAM-3) is a leukocyte-specific receptor involved in primary immune responses. We have investigated the interaction between ICAM-3 and ezrin/radixin/moesin (ERM) proteins and its role in LFA-1-induced cell-cell interactions and membrane positioning of ICAM-3 in polarized migrating lymphocytes. Protein-protein binding assays demonstrated a phosphatidylinositol 4,5-bisphosphate-induced association between ICAM-3 and the amino-terminal domain of ERM proteins. This interaction was not essential for the binding of ICAM-3 to LFA-1. Dynamic fluorescence videomicroscopy studies of cells demonstrated that moesin and ICAM-3 coordinately redistribute on the plasma membrane during lymphocyte migration. Furthermore, overexpression of the amino-terminal domain of moesin, which lacks the consensus moesin actin-binding site, caused the subcellular mislocalization of ICAM-3. A CD4 chimerical protein containing the cytoplasmic tail of ICAM-3 was targeted to the trailing edge. Point mutation of Ser(487), Ser(489), and Ser(496) to alanine in the juxtamembrane region of ICAM-3 significantly impaired both ERM binding and polarization of ICAM-3. ERM-directed polarization of ICAM-3 was also impaired by phosphorylation-like mutation of Ser(487) and Ser(489), but not of Ser(496). Our results underscore the key role of specific serine residues within the cytoplasmic region of ICAM-3 for its ERM-directed positioning at the trailing edge of motile lymphocytes.     

Ezrin/Radixin/Moesin (ERM) Proteins Bind to a Positively Charged Amino Acid Cluster in the Juxta-Membrane Cytoplasmic Domain of CD44, CD43, and ICAM-2

Abstract. CD44 has been identified as a membrane-binding partner for ezrin/radixin/moesin (ERM) proteins, plasma membrane/actin filament cross-linkers. ERM proteins, however, are not necessarily colocalized with CD44 in tissues, but with CD43 and ICAM-2 in some types of cells. We found that glutathione-S-transferase fusion proteins with the cytoplasmic domain of CD43 and ICAM-2, as well as CD44, bound to moesin in vitro. The regions responsible for the in vitro binding of CD43 and CD44 to moesin were narrowed down to their juxta-membrane 20–30–amino acid sequences in the cytoplasmic domain. These sequences and the cytoplasmic domain of ICAM-2 (28 amino acids) were all characterized by the positively charged amino acid clusters. When E-cadherin chimeric molecules bearing these positively charged amino acid clusters of CD44, CD43, or ICAM-2 were expressed in mouse L fibroblasts, they were co-concentrated with ERM proteins at microvilli, whereas those lacking these clusters were diffusely distributed on the cell surface. The specific binding of ERM proteins to the juxta-membrane positively charged amino acid clusters of CD44, CD43, and ICAM-2 was confirmed by immunoprecipitation and site-directed mutagenesis. From these findings, we conclude that ERM proteins bind to integral membrane proteins bearing a positively charged amino acid cluster in their juxta-membrane cytoplasmic domain.     

Roles of p-ERM and Rho-ROCK signaling in lymphocyte polarity and uropod formation

Front-rear asymmetry in motile cells is crucial for efficient directional movement. The uropod in migrating lymphocytes is a posterior protrusion in which several proteins, including CD44 and ezrin/radixin/moesin (ERM), are concentrated. In EL4.G8 T-lymphoma cells, Thr567 phosphorylation in the COOH-terminal domain of ezrin regulates the selective localization of ezrin in the uropod. Overexpression of the phosphorylation-mimetic T567D ezrin enhances uropod size and cell migration. T567D ezrin also induces construction of the CD44-associated polar cap, which covers the posterior cytoplasm in staurosporine-treated, uropod-disrupted EL4.G8 cells or in naturally unpolarized X63.653 myeloma cells in an actin cytoskeleton-dependent manner. Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor Y-27632 disrupts the uropod but not the polar cap, indicating that Rho-ROCK signaling is required for posterior protrusion but not for ERM phosphorylation. Phosphorylated ezrin associates with Dbl through its NH2-terminal domain and causes Rho activation. Moreover, constitutively active Q63L RhoA is selectively localized in the rear part of the cells. Thus, phosphorylated ERM has a potential function in establishing plasma membrane "posteriority" in the induction of the uropod in T lymphocytes.     

Differential expression of ezrin/radixin/moesin (ERM) and ERM-associated adhesion molecules in the blastocyst and uterus suggests their functions during implantation

Development of the blastocyst to implantation competency, differentiation of the uterus to the receptive state, and a cross talk between the implantation-competent blastocyst and the uterine luminal epithelium are all essential to the process of implantation. In the present investigation, we examined the possibility for a potential cross talk between the blastocyst and uterus involving the ezrin/radixin/moesin (ERM) proteins and ERM-associated cytoskeletal cross-linker proteins CD43, CD44, ICAM-1, and ICAM-2. In normal Day 4 blastocysts and after rendering dormant blastocysts to implantation-competent by estrogen in vivo (activated), the outer surface of mural trophectoderm cells showed much higher levels of radixin as compared to those in the polar trophectoderm cells, inner cell mass (ICM), and primitive endoderm. In contrast, ezrin was present on both the mural and the polar trophectoderm cell surfaces of normal Day 4 and activated blastocysts at higher intensity than dormant blastocysts. A distinct localization was noted in the primitive endoderm of dormant blastocysts that was not apparent in activated or normal Day 4 blastocysts. The expression of moesin was modestly higher at the mural trophectoderm of implantation-competent blastocysts, while the localization appeared to be present primarily on the polar trophectoderm cell surface of Day 4 blastocysts. The localization of ERM-associated adhesion molecules CD43, CD44, and ICAM-2 was more intense in the implantation-competent blastocysts compared with the dormant blastocysts. However, while CD44 was present both in the trophectoderm and in ICM, CD43 and ICAM-2 were localized primarily to the trophectoderm. The signal for ICAM-1 was very intense in the ICM but was modest in the trophectoderm. No significant changes in fluorescence intensity were noted between activated and dormant blastocysts. In the receptive uterus on Day 4 of pregnancy, ERM proteins were localized to the uterine epithelium, while on Day 5 the localization, especially of radixin and moesin, extended to the stroma surrounding the implantation chamber. With respect to ERM-associated adhesion molecules, while CD44 and ICAM-1 were exclusively localized in the stroma on Day 4, CD43 and ICAM-2 were localized to the epithelium. On Day 5, the localization of CD44 and ICAM-1 became highly concentrated in the antimesometrial stroma of the implantation chamber. The localization of CD43 and ICAM-2 remained mostly epithelial, although some stromal localization of CD43 was noted on Day 5. These results suggest that differential expression and distribution of ERM proteins and ERM-associated adhesion molecules are involved in the construction of the cellular architecture necessary for blastocyst activation and uterine receptivity leading to successful implantation.     

Nucleocapsid promotes localization of HIV-1 gag to uropods that participate in virological synapses between T cells

T cells adopt a polarized morphology in lymphoid organs, where cell-to-cell transmission of HIV-1 is likely frequent. However, despite the importance of understanding virus spread in vivo, little is known about the HIV-1 life cycle, particularly its late phase, in polarized T cells. Polarized T cells form two ends, the leading edge at the front and a protrusion called a uropod at the rear. Using multiple uropod markers, we observed that HIV-1 Gag localizes to the uropod in polarized T cells. Infected T cells formed contacts with uninfected target T cells preferentially via HIV-1 Gag-containing uropods compared to leading edges that lack plasma-membrane-associated Gag. Cell contacts enriched in Gag and CD4, which define the virological synapse (VS), are also enriched in uropod markers. These results indicate that Gag-laden uropods participate in the formation and/or structure of the VS, which likely plays a key role in cell-to-cell transmission of HIV-1. Consistent with this notion, a myosin light chain kinase inhibitor, which disrupts uropods, reduced virus particle transfer from infected T cells to target T cells. Mechanistically, we observed that Gag copatches with antibody-crosslinked uropod markers even in non-polarized cells, suggesting an association of Gag with uropod-specific microdomains that carry Gag to uropods. Finally, we determined that localization of Gag to the uropod depends on higher-order clustering driven by its NC domain. Taken together, these results support a model in which NC-dependent Gag accumulation to uropods establishes a preformed platform that later constitutes T-cell-T-cell contacts at which HIV-1 virus transfer occurs.     

Phosphorylated ERM is responsible for increased T cell polarization, adhesion, and migration in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune/inflammatory disease characterized by autoantibody production and abnormal T cells that infiltrate tissues through not well-known mechanisms. We report that SLE T lymphocytes display increased levels of CD44, ezrin, radixin, and moesin (ERM) phosphorylation, stronger actin polymerization, higher polar cap formation, and enhanced adhesion and chemotactic migration compared with T cells from patients with rheumatoid arthritis and normal individuals. Silencing of CD44 by CD44 small interfering RNA in SLE T cells inhibited significantly their ability to adhere and migrate as did treatment with Rho kinase and actin polymerization inhibitors. Forced expression of T567D-ezrin, a phosphorylation-mimic form, enhanced remarkably the adhesion and migration rate of normal T cells. Anti-CD3/TCR autoantibodies present in SLE sera caused increased ERM phosphorylation, adhesion, and migration in normal T cells. pERM and CD44 are highly expressed in T cells infiltrating in the kidneys of patients with lupus nephritis. These data prove that increased ERM phosphorylation represents a key molecular abnormality that guides T cell adhesion and migration in SLE patients.     

Microtubules Regulate Migratory Polarity through Rho/ROCK Signaling in T Cells

Background

Migrating leukocytes normally have a polarized morphology with an actin-rich lamellipodium at the front and a uropod at the rear. Microtubules (MTs) are required for persistent migration and chemotaxis, but how they affect cell polarity is not known.

Methodology/Principal Findings

Here we report that T cells treated with nocodazole to disrupt MTs are unable to form a stable uropod or lamellipodium, and instead often move by membrane blebbing with reduced migratory persistence. However, uropod-localized receptors and ezrin/radixin/moesin proteins still cluster in nocodazole-treated cells, indicating that MTs are required specifically for uropod stability. Nocodazole stimulates RhoA activity, and inhibition of the RhoA target ROCK allows nocodazole-treated cells to re-establish lamellipodia and uropods and persistent migratory polarity. ROCK inhibition decreases nocodazole-induced membrane blebbing and stabilizes MTs. The myosin inhibitor blebbistatin also stabilizes MTs, indicating that RhoA/ROCK act through myosin II to destabilize MTs.

Conclusions/Significance

Our results indicate that RhoA/ROCK signaling normally contributes to migration by affecting both actomyosin contractility and MT stability. We propose that regulation of MT stability and RhoA/ROCK activity is a mechanism to alter T-cell migratory behavior from lamellipodium-based persistent migration to bleb-based migration with frequent turning.     

Rac Activation by the T-Cell Receptor Inhibits T Cell Migration

Background

T cell migration is essential for immune responses and inflammation. Activation of the T-cell receptor (TCR) triggers a migration stop signal to facilitate interaction with antigen-presenting cells and cell retention at inflammatory sites, but the mechanisms responsible for this effect are not known.

Methodology/Principal Findings

Migrating T cells are polarized with a lamellipodium at the front and uropod at the rear. Here we show that transient TCR activation induces prolonged inhibition of T-cell migration. TCR pre-activation leads to cells with multiple lamellipodia and lacking a uropod even after removal of the TCR signal. A similar phenotype is induced by expression of constitutively active Rac1, and TCR signaling activates Rac1. TCR signaling acts via Rac to reduce phosphorylation of ezrin/radixin/moesin proteins, which are required for uropod formation, and to increase stathmin phosphorylation, which regulates microtubule stability. T cell polarity and migration is partially restored by inhibiting Rac or by expressing constitutively active moesin.

Conclusions/Significance

We propose that transient TCR signaling induces sustained inhibition of T cell migration via Rac1, increased stathmin phosphorylation and reduced ERM phosphorylation which act together to inhibit T-cell migratory polarity.     

Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 alpha-induced lymphocyte polarization and chemotaxis.

The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1 alpha receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1 alpha. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1 alpha-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.     

Immune synapse formation requires ZAP-70 recruitment by ezrin and CD43 removal by moesin

Immunological synapse (IS) formation involves receptor–ligand pair clustering and intracellular signaling molecule recruitment with a coincident removal of other membrane proteins away from the IS. As microfilament–membrane linkage is critical to this process, we investigated the involvement of ezrin and moesin, the two ezrin/radixin/moesin proteins expressed in T cells. We demonstrate that ezrin and moesin, which are generally believed to be functionally redundant, are differentially localized and have important and complementary functions in IS formation. Specifically, we find that ezrin directly interacts with and recruits the signaling kinase ZAP-70 to the IS. Furthermore, the activation of ezrin by phosphorylation is essential for this process. In contrast, moesin dephosphorylation and removal, along with CD43, are necessary to prepare a region of the cell cortex for IS. Thus, ezrin and moesin have distinct and critical functions in the T cell cortex during IS formation.     

Talin regulates moesin–NHE-1 recruitment to invadopodia and promotes mammary tumor metastasis

Invadopodia are actin-rich protrusions that degrade the extracellular matrix and are required for stromal invasion, intravasation, and metastasis. The role of the focal adhesion protein talin in regulating these structures is not known. Here, we demonstrate that talin is required for invadopodial matrix degradation and three-dimensional extracellular matrix invasion in metastatic breast cancer cells. The sodium/hydrogen exchanger 1 (NHE-1) is linked to the cytoskeleton by ezrin/radixin/moesin family proteins and is known to regulate invadopodium-mediated matrix degradation. We show that the talin C terminus binds directly to the moesin band 4.1 ERM (FERM) domain to recruit a moesin–NHE-1 complex to invadopodia. Silencing talin resulted in a decrease in cytosolic pH at invadopodia and blocked cofilin-dependent actin polymerization, leading to impaired invadopodium stability and matrix degradation. Furthermore, talin is required for mammary tumor cell motility, intravasation, and spontaneous lung metastasis in vivo. Thus, our findings provide a novel understanding of how intracellular pH is regulated and a molecular mechanism by which talin enhances tumor cell invasion and metastasis.     

Cutting edge: integration of human T lymphocyte cytoskeleton by the cytolinker plectin.

Chemokine-induced polarization of lymphocytes involves the rapid collapse of vimentin intermediate filaments (IFs) into an aggregate within the uropod. Little is known about the interactions of lymphocyte vimentin with other cytoskeletal elements. We demonstrate that human peripheral blood T lymphocytes express plectin, an IF-binding, cytoskeletal cross-linking protein. Plectin associates with a complex of structural proteins including vimentin, actin, fodrin, moesin, and lamin B in resting peripheral blood T lymphocytes. During chemokine-induced polarization, plectin redistributes to the uropod associated with vimentin and fodrin; their spatial distribution indicates that this vimentin-plectin-fodrin complex provides a continuous linkage from the nucleus (lamin B) to the cortical cytoskeleton. Overexpression of the plectin IF-binding domain in the T cell line Jurkat induces the perinuclear aggregation of vimentin IFs. Plectin is therefore likely to serve as an important organizer of the lymphocyte cytoskeleton and may regulate changes of lymphocyte cytoarchitecture during polarization and extravasation.     

Ezrin and Moesin Are Required for Efficient T Cell Adhesion and Homing to Lymphoid Organs

T cell trafficking between the blood and lymphoid organs is a complex, multistep process that requires several highly dynamic and coordinated changes in cyto-architecture. Members of the ezrin, radixin and moesin (ERM) family of actin-binding proteins have been implicated in several aspects of this process, but studies have yielded conflicting results. Using mice with a conditional deletion of ezrin in CD4+ cells and moesin-specific siRNA, we generated T cells lacking ERM proteins, and investigated the effect on specific events required for T cell trafficking. ERM-deficient T cells migrated normally in multiple in vitro and in vivo assays, and could undergo efficient diapedesis in vitro. However, these cells were impaired in their ability to adhere to the β1 integrin ligand fibronectin, and to polarize appropriately in response to fibronectin and VCAM-1 binding. This defect was specific for β1 integrins, as adhesion and polarization in response to ICAM-1 were normal. In vivo, ERM-deficient T cells showed defects in homing to lymphoid organs. Taken together, these results show that ERM proteins are largely dispensable for T cell chemotaxis, but are important for β1 integrin function and homing to lymphoid organs.     

High levels of ezrin expressed by human pancreatic adenocarcinoma cell lines with high metastatic potential.

Ezrin is a membrane cytoskeleton crosslinker protein that is a member of the ERM (ezrin/radixin/moesin) family. Ezrin binds adhesion molecules such as CD43, CD44, ICAM-1, and ICAM-2, which are implicated in cell migration and metastasis. Ezrin is expressed by many tumor cell lines; however, little is known about the function of ezrin in tumorigenesis and metastasis. Here, we investigated expression of ezrin in pancreatic adenocarcinoma cell lines of different metastatic potential. Among 16 pancreatic adenocarcinoma cell lines, several cell lines showed strong expression of ezrin. Two cell lines with high metastatic potential, S2-CP9 and S2-VP10, showed very high levels of ezrin mRNA and protein, whereas other sublines showed lower levels. There was no relationship between the expression levels of ezrin and the differentiation grades of the cell lines. These results suggest that there is a relationship between high expression of ezrin and metastatic potential of pancreatic carcinomas.     

Spatiotemporal regulation of moesin phosphorylation and rear release by Rho and serine/threonine phosphatase during neutrophil migration

Neutrophil motility is crucial to effective host defenses against microorganisms. While uropod retraction is a critical step in the migration of neutrophils, the underlying molecular mechanism is not well understood. Here, we show that inhibition of the Rho small GTPase with C3 exoenzyme prevented the retraction of trailing uropods, indicating that the process of rear release is mediated by a Rho signaling pathway. C3 exoenzyme caused marked elongation of directionally migrating neutrophils, suggesting an additional role for Rho in the maintenance of functional polarized cell shape. We also show that phosphorylation and dephosphorylation of the plasma membrane-actin filament cross-linker moesin are spatiotemporally controlled in migrating neutrophils. In particular, phosphorylation of moesin at threonine 558 depended on Rho activity. Videomicroscopy showed that dephosphorylation of this carboxy-terminal threonine preceded uropod retraction. Calyculin A, an inhibitor of type 1 and type 2A serine/threonine phosphatases, suppressed the moesin dephosphorylation and impaired uropod retraction in a dose-dependent manner. Cypermethrin, an inhibitor of type 2B serine/threonine phosphatase, had no such effects. The finding that Rho small GTPase and type 1/type 2A phosphatases are involved in rear release yields novel insights into the biochemical mechanisms of neutrophil migration.     

Cytoskeleton-dependent Membrane Domain Segregation during Neutrophil Polarization

On treatment with chemoattractant, the neutrophil plasma membrane becomes organized into detergent-resistant membrane domains (DRMs), the distribution of which is intimately correlated with cell polarization. Plasma membrane at the front of polarized cells is susceptible to extraction by cold Triton X-100, whereas membrane at the rear is resistant to extraction. After cold Triton X-100 extraction, DRM components, including the transmembrane proteins CD44 and CD43, the GPI-linked CD16, and the lipid analog, DiIC(16), are retained within uropods and cell bodies. Furthermore, CD44 and CD43 interact concomitantly with DRMs and with the F-actin cytoskeleton, suggesting a mechanism for the formation and stabilization of DRMs. By tracking the distribution of DRMs during polarization, we demonstrate that DRMs progress from a uniform distribution in unstimulated cells to small, discrete patches immediately after activation. Within 1 min, DRMs form a large cap comprising the cell body and uropod. This process is dependent on myosin in that an inhibitor of myosin light chain kinase can arrest DRM reorganization and cell polarization. Colabeling DRMs and F-actin revealed a correlation between DRM distribution and F-actin remodeling, suggesting that plasma membrane organization may orient signaling events that control cytoskeletal rearrangements and, consequently, cell polarity.     

Structural basis for CD44 recognition by ERM proteins

CD44 is an important adhesion molecule that functions as the major hyaluronan receptor which mediates cell adhesion and migration in a variety of physiological and pathological processes. Although full activity of CD44 requires binding to ERM (ezrin/radixin/moesin) proteins, the CD44 cytoplasmic region, consisting of 72 amino acid residues, lacks the Motif-1 consensus sequence for ERM binding found in intercellular adhesion molecule (ICAM)-2 and other adhesion molecules of the immunoglobulin superfamily. Ultracentrifugation sedimentation studies and circular dichroism measurements revealed an extended monomeric form of the cytoplasmic peptide in solution. The crystal structure of the radixin FERM domain complexed with a CD44 cytoplasmic peptide reveals that the KKKLVIN sequence of the peptide forms a beta strand followed by a short loop structure that binds subdomain C of the FERM domain. Like Motif-1 binding, the CD44 beta strand binds the shallow groove between strand beta5C and helix alpha1C and augments the beta sheet beta5C-beta7C from subdomain C. Two hydrophobic CD44 residues, Leu and Ile, are docked into a hydrophobic pocket with the formation of hydrogen bonds between Asn of the CD44 short loop and loop beta4C-beta5C from subdomain C. This binding mode resembles that of NEP (neutral endopeptidase 24.11) rather than ICAM-2. Our results reveal a characteristic versatility of peptide recognition by the FERM domains from ERM proteins, suggest a possible mechanism by which the CD44 tail is released from the cytoskeleton for nuclear translocation by regulated intramembrane proteolysis, and provide a structural basis for Smad1 interactions with activated CD44 bound to ERM protein.