Lipid Raft Colocalization Research Articles

Piezo1 Mechano-Activation Is Augmented by Resveratrol and Differs between Colorectal Cancer Cells of Primary and Metastatic Origin.

Cancer cells must survive aberrant fluid shear stress (FSS) in the circulation to metastasize. Herein, we investigate the role that FSS has on colorectal cancer cell apoptosis, proliferation, membrane damage, calcium influx, and therapeutic sensitization. We tested this using SW480 (primary tumor) and SW620 cells (lymph node metastasis) derived from the same patient. The cells were exposed to either shear pulses, modeling millisecond intervals of high FSS seen in regions of turbulent flow, or sustained shear to model average magnitudes experienced by circulating tumor cells. SW480 cells were significantly more sensitive to FSS-induced death than their metastatic counterparts. Shear pulses caused significant cell membrane damage, while constant shear decreased cell proliferation and increased the expression of CD133. To investigate the role of mechanosensitive ion channels, we treated cells with the Piezo1 agonist Yoda1, which increased intracellular calcium. Pretreatment with resveratrol further increased the calcium influx via the lipid-raft colocalization of Piezo1. However, minimal changes in apoptosis were observed due to calcium saturation, as predicted via a computational model of apoptosis. Furthermore, SW480 cells had increased levels of Piezo1, calcium influx, and TRAIL-mediated apoptosis compared to SW620 cells, highlighting differences in the mechano-activation of metastatic cells, which may be a necessary element for successful dissemination in vivo.

Morphologies of Charged Diblock Copolymers Simulated with a Neutral Coarse-Grained Model

We present the results of coarse grained molecular dynamics simulation using a charge free model that is able to capture different regions of the morphological phase diagram of charged diblock copolymers. Specifically, we were able to reproduce many phases of the poly(acrylic acid)-(1,4)-polybutadiene (PAA-PBA) diblock copolymer, Ca(2+) and water systems as a function of pH and calcium concentration with short-range LJ type potentials. The morphologies observed range from bilayers to cylinders to spherical micelles. Such polyanionic/cationic amphiphiles in water typically present multiple challenges for molecular simulations, particularly due to the many charge interactions that are long ranged and computationally costly. Further, it is precisely these interactions that are thought to modulate large amphiphile assemblies of interest such as lipid rafts. However, our model is able to reproduce different morphologies due to pH and with or without the addition of Ca(2+) as well as the lateral phase segregation and the domain registration observed in neutral and charged diblock copolymer mixtures. The results suggest that the overall effect of charges is a local structural rearrangement that renormalizes the steric repulsion between the headgroups. This simple model, which is devoid of charges, is able to reproduce the complex phase diagram and can be used to investigate collective phenomena in these charged systems such as domain formation and registration or colocalization of lipid rafts across bilayer leaflets.

The insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) alters the membrane raft location of the TSH receptor stably expressed in Chinese hamster ovary cells

DDT is a highly lipophilic molecule known to deplete membrane rafts of their phosphoglycolipid and cholesterol contents. However, we have recently shown that DDT can also alter the thyroid homeostasis by inhibiting TSH receptor (TSHr) internalization. The present study was undertaken to verify whether DDT goitrogenic effects are due to the insecticide acting directly on TSHr or via alteration of the membrane rafts hosting the receptor itself. Our results demonstrate that, in CHO-TSHr transfected cells, TSHr is activated in the presence of TSH, while it is inhibited following DDT exposure. DDT can also reduce the endocytic vesicular traffic, alter the extension of multi-branched microvilli along their plasma membranes and induce TSHr shedding in vesicular forms. To verify whether TSHr displacement might depend on DDT altering the raft constitution of CHO-TSHr cell membranes the extent of TSHr and lipid raft co-localization was examined by confocal microscopy. Evidence shows that receptor/raft co-localization increased significantly upon exposure to TSH, while receptors and lipid rafts become dislodged on opposite cell poles in DDT-exposed CHO-TSHr cells. As a control, under similar culturing conditions, diphenylethylene, which is known to be a lipophilic substance that is structurally related to DDT, did not affect the extent of TSHr and lipid raft co-localization in CHO-TSHr cells treated with TSH. These findings corroborate and extend our view that, in CHO cells, the DDT disrupting action on TSHr is primarily due to the insecticide acting on membranes to deplete their raft cholesterol content, and that the resulting inhibition on TSHr internalization is due to receptor dislodgement from altered raft microdomains of the plasma membrane.

Ethanol Inhibits Lipid Raft-Mediated TCR Signaling and IL-2 Expression: Potential Mechanism of Alcohol-Induced Immune Suppression

Alcohol abuse has long-term deleterious effects on the immune system, and results in a depletion and loss of function of CD4(+) T lymphocytes, which regulate both innate and adaptive immunity. T-lymphocyte activation via T-cell receptor (TCR) involves the lipid raft colocalization and aggregation of proteins into the immunological signalosome, which triggers a signaling cascade resulting in the production of interleukin-2 (IL-2). IL-2 regulates the proliferation and clonal expansion of activated T cells and is essential for an effective immune response. The present work examines the mechanisms underlying ethanol-induced dysfunction of CD4(+) T lymphocytes based on the hypothesis that ethanol downregulates lipid raft-mediated TCR signal transduction and resultant IL-2 production. Primary or cultured human T lymphocytes were exposed to ethanol for 24 hours prior to stimulation with anti-CD3/anti-CD28 antibodies or phytohemagglutinin. Effects of ethanol exposure on TCR-signaling (including activation of Lck, ZAP70, LAT, and PLCγ1) and IL-2 gene expression were examined. Exposure of both primary and cultured human CD4(+) T lymphocytes to physiologically relevant concentrations of ethanol leads to down-regulation of IL-2 mRNA and protein via inhibition of DNA-binding activity of NFAT, the essential transcription factor for IL-2. Ethanol decreases tyrosine phosphorylation and activation of upstream signaling proteins PLCγ1, LAT, ZAP70, and Lck. These effects are prevented by inhibition of metabolism of ethanol. Sucrose density gradient fractionation and confocal microscopy revealed that ethanol inhibited essential upstream lipid raft-mediated TCR-dependent signaling events, namely colocalization of Lck, ZAP70, LAT, and PLCγ1 with plasma membrane lipid rafts. Overall, our data demonstrate that ethanol inhibits lipid raft-mediated TCR-signaling in CD4(+) T lymphocytes, resulting in suppression of IL-2 production. These findings may represent a novel mechanism underlying alcohol abuse-associated immune suppression and may be particularly relevant in diseases such as HIV/AIDS and hepatitis C virus infection where alcohol abuse is a known comorbidity.

Hyaluronan fragments contribute to the ozone-primed immune response to lipopolysaccharide.

Hyaluronan is a high-molecular mass component of pulmonary extracelluar matrix, and lung injury can generate a low-molecular mass hyaluronan (HA) fragment that functions as endogenous ligand to cell surface receptors CD44 and TLR4. This leads to activation of intracellular NF-κB signaling and proinflammatory cytokine production. Based on previous information that ozone exposure causes increased HA in bronchial alveolar lavage fluid and ozone pre-exposure primes immune response to inhaled LPS, we hypothesized that HA production during ozone exposure augments the inflammatory response to LPS. We demonstrate that acute ozone exposure at 1 part per million for 3 h primes the immune response to low-dose aerosolized LPS in C57BL/6J mice, resulting in increased neutrophil recruitment into the airspaces, increased levels of protein and proinflammatory cytokines in the bronchoalveolar lavage fluid, and increased airway hyperresponsiveness. Intratracheal instillation of endotoxin-free HA (25 μg) enhances the biological response to inhaled LPS in a manner similar to ozone pre-exposure. In vitro studies using bone marrow-derived macrophages indicate that HA enhances LPS responses measured by TNF-α production, while immunofluorescence staining of murine alveolar macrophages demonstrates that HA induces TLR4 peripheralization and lipid raft colocalization. Collectively, our observations support that ozone primes macrophage responsiveness to low-dose LPS, in part, due to HA-induced TLR4 peripheralization in lung macrophages.

Ethanol Inhibits Lipid Raft‐Dependent TCR Signaling and IL‐2 Expression: Potential Mechanism of Alcohol‐Induced Immune Suppression

Alcohol abuse has long‐term deleterious effects on the immune system, and results in reduced function of CD4+T lymphocytes, which regulate both innate and adaptive immunity. T lymphocyte activation via T‐cell receptor (TCR) involves the plasma membrane lipid raft‐dependent aggregation of proteins into the immunological signalosome, which in turn, triggers a signaling cascade resulting in interleukin‐2 (IL‐2) production. IL‐2 is a critical cytokine that regulates the proliferation/clonal expansion of activated T‐cells, and is essential for an effective immune response.The present work examines the mechanisms underlying ethanol‐induced dysfunction of CD4+T lymphocytes, and demonstrates for the first time, that physiologically relevant concentrations of ethanol inhibit lipid raft‐dependent TCR signaling and decrease production of IL‐2 in both primary and cultured human CD4+T lymphocytes. Specifically, ethanol substantially down‐regulates lipid raft colocalization and activation/phosphorylation of Lck, ZAP70 and LAT, resulting in a marked decrease in DNA‐binding activity of NFAT and IL‐2 mRNA and protein. Importantly, inhibition of ethanol metabolism prevented these inhibitory effects. Overall, our findings have clinical relevance in alcohol abuse‐associated immune dysfunction, especially in conditions such as HIV and HCV infection where alcohol abuse is a known comorbidity.

Granulocyte-Derived Cationic Peptides (GDCPs) Present in Leucophoresis Products Enhance Homing of Hematopoietic Stem Cells (HSCs) to SDF-1 Gradient; Potential Implications for Accelerated Recovery of Hematopoiesis After Transplantation of Mobilized Peripheral Blood Stem Cells (PBSC).

Abstract 371▪▪This icon denotes an abstract that is clinically relevant.Current strategies to accelerate hematopoietic reconstitution after transplantation, include transplantation of greater numbers of HSC or ex vivo expansion of harvested HSC before transplant. However, the number of HSC availabel for allogeneic or autologous transplantation can be low (e.g., umbilical cord blood, poor mobilizers) and strategies to expand HSC and maintain equivalent engraftment capability ex vivo are limited. We reported that some compounds present in leucopheresis products [(e.g., platelet-derived microparticles (Blood 2001, 98: 3143)] and some complement cascade cleavage fragments, e.g., anaphylatoxin C3a (Blood 2005, 101, 3784), enhance the homing responses of HSC to SDF-1 gradient. We recently noted that small cationic peptides released from activated granulocytes (beta2-defensin and cathelicidin) positively prime responsiveness of murine and human HSC to SDF-1 gradient (Leukemia 2009; in press). Accordingly, both compounds enhanced transwell migration of HSC to low threshold doses of SDF-1. This phenomenon was not receptor-dependent, as agonists of membrane receptors that may bind beta2-defensin (FPRL-1), cathelicidin (CCR6) - FPRL-1 agonist, and MIP-3alpha, respectively, did not show similar priming effects. This could be explained by affected distribution of membrane lipids by cationic peptides. In support of this notion, an inhibitor of cell membrane raft formation (methyl-b-cyclodextran) inhibited the priming effect of both compounds, indicating this effect is dependent on CXCR4 incorporation into lipid rafts. Direct confocal analysis of CXCR4 and lipid raft colocalization in the presence or absence of cationic peptides confirmed these findings. Because leucopheresis products are enriched in activated granulocytes that release beta2-defensin and cathelicidin, we tested whether this may explain why mobilized peripheral blood stem cells (PBSC) engraft faster compared to HSC isolated directly from bone marrow (BM) in a murine BM transplant model. Accordingly, syngeneic BMMNCs were exposed ex vivo to beta2-defensin or cathelicidin for 30 minutes and subsequently transplanted into lethally irradiated recipients. We noted that animals transplanted with BM cells primed by those cationic peptides showed accelerated recovery of platelets and neutrophils by ∼3-5 days compared to unprimed control cells. We envision that small cationic peptides, which primarily possess antimicrobial functions and are harmless to mammalian cells, could be clinically applied to prime human HSC before transplantation. This novel approach would be particularly important in cord blood transplantation, where the number of HSC availabel for transplantation is usually limited. We postulate that this promising strategy warrants further investigations. Disclosures:No relevant conflicts of interest to declare.

Visualizing lipid raft dynamics and early signaling events during antigen receptor-mediated B-lymphocyte activation.

Recent biochemical evidence indicates that an early event in signal transduction by the B-cell antigen receptor (BCR) is its translocation to specialized membrane subdomains known as lipid rafts. We have taken a microscopic approach to image lipid rafts and early events associated with BCR signal transduction. Lipid rafts were visualized on primary splenic B lymphocytes from wild-type or anti-hen egg lysozyme BCR transgenic mice, and on a mature mouse B-cell line Bal 17 by using fluorescent conjugates of cholera toxin B subunit or a Lyn-based chimeric protein, which targets green fluorescent protein to the lipid raft compartment. Time-lapse imaging of B cells stimulated via the BCR with the antigen hen egg lysozyme, or surrogate for antigen anti-IgM, demonstrated that lipid rafts are highly dynamic entities, which move laterally on the surface of these cells and coalesce into large regions. These regions of aggregated lipid rafts colocalized with the BCR and tyrosine-phosphorylated proteins. Microscopic imaging of live B cells also revealed an inducible colocalization of lipid rafts with the tyrosine kinase Syk and the receptor tyrosine phosphatase CD45. These two proteins play indispensable roles in BCR-mediated signaling but are not detectable in biochemically purified lipid raft fractions. Strikingly, BCR stimulation also induced the formation of long, thread-like filopodial projections, similar to previously described structures called cytonemes. These B-cell cytonemes are rich in lipid rafts and actin filaments, suggesting that they might play a role in long-range communication and/or transportation of signaling molecules during an immune response. These results provide a window into the morphological and molecular organization of the B-cell membrane during the early phase of BCR signaling.