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

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

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

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

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

Hypogonadism associated with muscle atrophy, physical inactivity and ESA hyporesponsiveness in men undergoing haemodialysis

AbstractBackgroundTestosterone deficiency (hypogonadism) is common among men undergoing haemodialysis, but its clinical implications are not well characterized. Testosterone is an anabolic hormone that induces erythrocytosis and muscle synthesis. We hypothesized that testosterone deficiency would be associated with low muscle mass, physical inactivity and higher dosages of erythropoietin-stimulating agents (ESA).MethodsSingle-center cross-sectional study of 57 male haemodialysis patients. None of the patients was undergoing testosterone replacement therapy. Total testosterone was measured in serum. Body composition (by bioelectrical impedance analysis) and physical activity (by the use of pedometers) were assessed. Patients with testosterone levels below the normal range were considered hypogonadal.ResultsMean testosterone level was 321±146ng/dL; 20 patients (35%) were hypogonadal. Hypogonadal patients were older and had lower mean arterial blood pressure, higher interleukin-6 levels, lower lean body mass and higher fat body mass. A negative association between testosterone and normalized ESA dose was found in uni- and multivariate regression analyses. Testosterone levels directly correlated with lean body mass regardless of confounders. Hypogonadal patients had lower physical activity than their counterparts [2753±1784 vs. 4291±3225steps/day (p=0.04)]. The relationship between testosterone and physical activity was independent of age, comorbidities and inflammatory markers, but dependent on the proportion of muscle mass.ConclusionHypogonadism is common in our male haemodialysis population and is associated with higher ESA doses, reduced muscle mass and lower physical activity. The link between low testosterone levels and physical inactivity may conceivably relate to reduced muscle mass due to inadequate muscle protein synthesis

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

Adolescents and young adults evaluating a website for affective‐sexual information and education: a mixed‐methods study protocol

The website Sexe Joves is a website on sexuality of the Department of Health of the Government of Catalonia (Spain). This study aims to understand the experiences and opinions of people aged 14 to 25 regarding this website, taking into account sex, gender identity, sexual orientation, socioeconomic status and location within Catalonia (urban, semiurban and rural areas). With the objective of improving the website and adpating the resources allocated to it, this study evaluates whether this population is familiar with it and uses it, as well as the website's usability and accessibility (digital equity), usefulness and the relevance of its content. A parallel convergent triangulation design is used: a qualitative study using a social constructivist perspective, and an observational, descriptive and cross-sectional quantitative study. We conduct a discourse analysis of participants and use an 'ad hoc' questionnaire to collect quantitative data. A descriptive analysis of all variables is carried out. Affective-sexual education aimed at young people must stem from their participation and the whole range of sexual and gender diversity in order to reach the entire population equally. This analysis will contribute to the design of new strategies for the wesbite Sexe Joves, a public health resource, in order to improve affective-sexual education for young people

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

Statins Inhibit HIV-1 Infection by Down-regulating Rho Activity

Human immunodeficiency virus (HIV)-1 infectivity requires actin-dependent clustering of host lipid raft–associated receptors, a process that might be linked to Rho guanosine triphosphatase (GTPase) activation. Rho GTPase activity can be negatively regulated by statins, a family of drugs used to treat hypercholesterolemia in man. Statins mediate inhibition of Rho GTPases by impeding prenylation of small G proteins through blockade of 3-hydroxy-3-methylglutaryl coenzyme A reductase. We show that statins decreased viral load and increased CD4+ cell counts in acute infection models and in chronically HIV-1–infected patients. Viral entry and exit was reduced in statin-treated cells, and inhibition was blocked by the addition of l-mevalonate or of geranylgeranylpyrophosphate, but not by cholesterol. Cell treatment with a geranylgeranyl transferase inhibitor, but not a farnesyl transferase inhibitor, specifically inhibited entry of HIV-1–pseudotyped viruses. Statins blocked Rho-A activation induced by HIV-1 binding to target cells, and expression of the dominant negative mutant RhoN19 inhibited HIV-1 envelope fusion with target cell membranes, reducing cell infection rates. We suggest that statins have direct anti–HIV-1 effects by targeting Rho