Orchestration of lymphocyte chemotaxis by mitochondrial dynamics

Lymphocyte traffic is required to maintain homeostasis and perform appropriate immunological reactions. To migrate into inflamed tissues, lymphocytes must acquire spatial and functional asymmetries. Mitochondria are highly dynamic organelles that distribute in the cytoplasm to meet specific cellular needs, but whether this is essential to lymphocyte functions is unknown. We show that mitochondria specifically concentrate at the uropod during lymphocyte migration by a process involving rearrangements of their shape. Mitochondrial fission facilitates relocation of the organelles and promotes lymphocyte chemotaxis, whereas mitochondrial fusion inhibits both processes. Our data substantiate a new role for mitochondrial dynamics and suggest that mitochondria redistribution is required to regulate the motor of migrating cells

Specific SHP-2 partitioning in raft domains triggers integrin-mediated signaling via Rho activation

Copyright © by The Rockefeller University PressCell signaling does not occur randomly over the cell surface, but is integrated within cholesterol-enriched membrane domains, termed rafts. By targeting SHP-2 to raft domains or to a non-raft plasma membrane fraction, we studied the functional role of rafts in signaling. Serumdepleted, nonattached cells expressing the raft SHP-2 form, but not non-raft SHP-2, display signaling events resembling those observed after fibronectin attachment, such as 1 integrin clustering, 397 Y-FAK phosphorylation, and ERK activation, and also increases Rho-GTP levels. Expression of C the dominant negative N19Rho abrogates raft-SHP-2–induced signaling, suggesting that Rho activation is a downstream event in SHP-2 signaling. Expression of a catalytic inactive SHP-2 mutant abrogates the adhesion-induced feedback inhibition of Rho activity, suggesting that SHP-2 contributes to adhesion-induced suppression of Rho activity. Because raft recruitment of SHP-2 occurs physiologically after cell attachment, these results provide a mechanism by which SHP-2 may influence cell adhesion and migration by spatially regulating Rho activity.Peer reviewe

Type I phosphatidylinositol 4-phosphate 5-kinase controls neutrophil polarity and directional movement

Directional cell movement in response to external chemical gradients requires establishment of front–rear asymmetry, which distinguishes an up-gradient protrusive leading edge, where Rac-induced F-actin polymerization takes place, and a down-gradient retractile tail (uropod in leukocytes), where RhoA-mediated actomyosin contraction occurs. The signals that govern this spatial and functional asymmetry are not entirely understood. We show that the human type I phosphatidylinositol 4-phosphate 5-kinase isoform β (PIPKIβ) has a role in organizing signaling at the cell rear. We found that PIPKIβ polarized at the uropod of neutrophil-differentiated HL60 cells. PIPKIβ localization was independent of its lipid kinase activity, but required the 83 C-terminal amino acids, which are not homologous to other PIPKI isoforms. The PIPKIβ C terminus interacted with EBP50 (4.1-ezrin-radixin-moesin (ERM)-binding phosphoprotein 50), which enabled further interactions with ERM proteins and the Rho-GDP dissociation inhibitor (RhoGDI). Knockdown of PIPKIβ with siRNA inhibited cell polarization and impaired cell directionality during dHL60 chemotaxis, suggesting a role for PIPKIβ in these processes

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 γ 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

Vacancy induced zero energy modes in graphene stacks: The case of ABC trilayer

The zero energy modes induced by vacancies in ABC stacked trilayer graphene are investigated. Depending on the position of the vacancy, a new zero energy solution is realised, different from those obtained in multilayer compounds with Bernal stacking. The electronic modification induced in the sample by the new vacancy states is characterised by computing the local density of states and their localisation properties are studied by the inverse participation ratio. We also analyse the situation in the presence of a gap in the spectrum due to a perpendicular electric field.Comment: 6 pages, 4 figures Published in special issue: Exploring Graphene, Recent Research Advance

Blocking of HIV-1 Infection by Targeting CD4 to Nonraft Membrane Domains

Human immunodeficiency virus (HIV)-1 infection depends on multiple lateral interactions between the viral envelope and host cell receptors. Previous studies have suggested that these interactions are possible because HIV-1 receptors CD4, CXCR4, and CCR5 partition in cholesterol-enriched membrane raft domains. We generated CD4 partitioning mutants by substituting or deleting CD4 transmembrane and cytoplasmic domains and the CD4 ectodomain was unaltered. We report that all CD4 mutants that retain raft partitioning mediate HIV-1 entry and CD4-induced Lck activation independently of their transmembrane and cytoplasmic domains. Conversely, CD4 ectodomain targeting to a nonraft membrane fraction results in a CD4 receptor with severely diminished capacity to mediate Lck activation or HIV-1 entry, although this mutant binds gp120 as well as CD4wt. In addition, the nonraft CD4 mutant inhibits HIV-1 X4 and R5 entry in a CD4+ cell line. These results not only indicate that HIV-1 exploits host membrane raft domains as cell entry sites, but also suggest new strategies for preventing HIV-1 infection

CCR5 Expression Influences the Progression of Human Breast Cancer in a p53-dependent Manner

Chemokines are implicated in tumor pathogenesis, although it is unclear whether they affect human cancer progression positively or negatively. We found that activation of the chemokine receptor CCR5 regulates p53 transcriptional activity in breast cancer cells through pertussis toxin–, JAK2-, and p38 mitogen–activated protein kinase–dependent mechanisms. CCR5 blockade significantly enhanced proliferation of xenografts from tumor cells bearing wild-type p53, but did not affect proliferation of tumor xenografts bearing a p53 mutation. In parallel, data obtained in a primary breast cancer clinical series showed that disease-free survival was shorter in individuals bearing the CCR5Δ32 allele than in CCR5 wild-type patients, but only for those whose tumors expressed wild-type p53. These findings suggest that CCR5 activity influences human breast cancer progression in a p53-dependent manner

Specific SHP-2 partitioning in raft domains triggers integrin-mediated signaling via Rho activation

Cell signaling does not occur randomly over the cell surface, but is integrated within cholesterol-enriched membrane domains, termed rafts. By targeting SHP-2 to raft domains or to a non-raft plasma membrane fraction, we studied the functional role of rafts in signaling. Serum-depleted, nonattached cells expressing the raft SHP-2 form, but not non-raft SHP-2, display signaling events resembling those observed after fibronectin attachment, such as β(1) integrin clustering, (397)Y-FAK phosphorylation, and ERK activation, and also increases Rho-GTP levels. Expression of the dominant negative N19Rho abrogates raft-SHP-2–induced signaling, suggesting that Rho activation is a downstream event in SHP-2 signaling. Expression of a catalytic inactive SHP-2 mutant abrogates the adhesion-induced feedback inhibition of Rho activity, suggesting that SHP-2 contributes to adhesion-induced suppression of Rho activity. Because raft recruitment of SHP-2 occurs physiologically after cell attachment, these results provide a mechanism by which SHP-2 may influence cell adhesion and migration by spatially regulating Rho activity

Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: ErbB2-positive breast cancer is characterized by highly aggressive phenotypes and reduced responsiveness to standard therapies. Although specific ErbB2-targeted therapies have been designed, only a small percentage of patients respond to these treatments and most of them eventually relapse. The existence of this population of particularly aggressive and non-responding or relapsing patients urges the search for novel therapies. The purpose of this study was to determine whether cannabinoids might constitute a new therapeutic tool for the treatment of ErbB2-positive breast tumors. We analyzed their antitumor potential in a well established and clinically relevant model of ErbB2-driven metastatic breast cancer: the MMTV-neu mouse. We also analyzed the expression of cannabinoid targets in a series of 87 human breast tumors. RESULTS: Our results show that both Delta9-tetrahydrocannabinol, the most abundant and potent cannabinoid in marijuana, and JWH-133, a non-psychotropic CB2 receptor-selective agonist, reduce tumor growth, tumor number, and the amount/severity of lung metastases in MMTV-neu mice. Histological analyses of the tumors revealed that cannabinoids inhibit cancer cell proliferation, induce cancer cell apoptosis, and impair tumor angiogenesis. Cannabinoid antitumoral action relies, at least partially, on the inhibition of the pro-tumorigenic Akt pathway. We also found that 91% of ErbB2-positive tumors express the non-psychotropic cannabinoid receptor CB2. CONCLUSIONS: Taken together, these results provide a strong preclinical evidence for the use of cannabinoid-based therapies for the management of ErbB2-positive breast cancer

Superoxide Dismutase-3 Downregulates Laminin α5 Expression in Tumor Endothelial Cells via the Inhibition of Nuclear Factor Kappa B Signaling.

The balance between laminin isoforms containing the α5 or the α4 chain in the endothelial basement membrane determines the site of leukocyte diapedesis under inflammatory conditions. Extracellular superoxide dismutase (SOD3) induces laminin α4 expression in tumor blood vessels, which is associated with enhanced intratumor T cell infiltration in primary human cancers. We show now that SOD3 overexpression in neoplastic and endothelial cells (ECs) reduces laminin α5 in tumor blood vessels. SOD3 represses the laminin α5 gene (LAMA5), but LAMA5 expression is not changed in SOD1-overexpressing cells. Transcriptomic analyses revealed SOD3 overexpression to change the transcription of 1682 genes in ECs, with the canonical and non-canonical NF-κB pathways as the major SOD3 targets. Indeed, SOD3 reduced the transcription of well-known NF-κB target genes as well as NF-κB-driven promoter activity in ECs stimulated with tumor necrosis factor (TNF)-α, an NF-κB signaling inducer. SOD3 inhibited the phosphorylation and degradation of IκBα (nuclear factor of the kappa light polypeptide gene enhancer in B-cells inhibitor alpha), an NF-κB inhibitor. Finally, TNF-α was found to be a transcriptional activator of LAMA5 but not of LAMA4; LAMA5 induction was prevented by SOD3. In conclusion, SOD3 is a major regulator of laminin balance in the basement membrane of tumor ECs, with potential implications for immune cell infiltration into tumors