Sticholysin I–membrane interaction: An interplay between the presence of sphingomyelin and membrane fluidity

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer.     

The formation of ordered nanoclusters controls cadherin anchoring to actin and cell–cell contact fluidity

Oligomerization of cadherins could provide the stability to ensure tissue cohesion. Cadherins mediate cell–cell adhesion by forming trans-interactions. They form cis-interactions whose role could be essential to stabilize intercellular junctions by shifting cadherin clusters from a fluid to an ordered phase. However, no evidence has been provided so far for cadherin oligomerization in cellulo and for its impact on cell–cell contact stability. Visualizing single cadherins within cell membrane at a nanometric resolution, we show that E-cadherins arrange in ordered clusters, providing the first demonstration of the existence of oligomeric cadherins at cell–cell contacts. Studying the consequences of the disruption of the cis-interface, we show that it is not essential for adherens junction formation. Its disruption, however, increased the mobility of junctional E-cadherin. This destabilization strongly affected E-cadherin anchoring to actin and cell–cell rearrangement during collective cell migration, indicating that the formation of oligomeric clusters controls the anchoring of cadherin to actin and cell–cell contact fluidity.     

Thyroid hormones increase Na+–Pi co-transport activity in intestinal brush border membrane: Role of membrane lipid composition and fluidity

In the present study, we documented the promising role of thyroid hormones status in animals in modulation of Na⁺–P_i transport activity in intestinal brush border membrane vesicles (BBMV) which was accompanied with alterations in BBM lipid composition and fluidity. Augmentation of net P_i balance in hyperthyroid (Hyper-T) rats was fraternized with accretion of P_i transport across BBMV isolated from intestine of Hyper-T rats as compared to hypothyroid (Hypo-T) and euthyroid (Eu-T) rats while Na⁺–P_i transport across BBMV was decreased in Hypo-T rats relative to Eu-T rats. Increment in Na⁺–P_i transport in intestinal BBMV isolated from Hyper-T rats was manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport system. Furthermore, BBMV lipid composition profile in intestinal BBM from Hyper-T was altered to that of Hypo-T rats and Eu-T rats. The molar ratio of cholesterol/phospholipids was higher in intestinal BBM from Hypo-T rats. Fluorescence anistropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the intestinal BBM of Hyper-T rats and decreased in Hypo-T rats as compared to Eu-T rats which corroborated with the alteration in membrane fluidity in response to thyroid hormone status of animals. Therefore, thyroid hormone mediated change in membrane fluidity might play an important role in modulating Na⁺–P_i transport activity of intestinal BBM. (Mol Cell Biochem 278: 195–202, 2005)     

Palmitate and oleate modify membrane fluidity and kinase activities of INS-1E β-cells alongside altered metabolism-secretion coupling

Chronic exposure to elevated levels of glucose and free fatty acids impairs beta-cell function, leading to insulin secretion defects and eventually beta-cell failure. Using a semi-high throughput approach applied to INS-1E beta-cells, we tested multiple conditions of chronic exposure to basal, intermediate and high glucose, combined with saturated versus mono- and polyunsaturated fatty acids in order to assess cell integrity, lipid metabolism, mitochondrial function, glucose-stimulated calcium rise and secretory kinetics. INS-1E beta-cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mM) without or with BSA-complexed 0.4 mM saturated (C16:0 palmitate), monounsaturated (C18:1 oleate) or polyunsaturated (C18:2 linoleate, C18:3 linolenate) fatty acids, resulting in 0.1–0.5 μM unbound fatty acids. Accumulation of triglycerides in cells exposed to fatty acids was glucose-dependent, oleate inducing the strongest lipid storage and protecting against glucose-induced cytotoxicity. The combined chronic exposure to both high glucose and either palmitate or oleate altered mitochondrial function as well as glucose-induced calcium rise. This pattern did not directly translate at the secretory level since palmitate and oleate exhibited distinct effects on the first and the second phases of glucose-stimulated exocytosis. Both fatty acids changed the activity of kinases, such as the MODY-associated BLK. Additionally, chronic exposure to fatty acids modified membrane physicochemical properties by increasing membrane fluidity, oleate exhibiting larger effects compared to palmitate. Chronic fatty acids differentially and specifically exacerbated some of the glucotoxic effects, without promoting cytotoxicity on their own. Each of the tested fatty acids functionally modified INS-1E beta-cell, oleate inducing the strongest effects.     

Vpu Antagonizes BST-2–Mediated Restriction of HIV-1 Release via β-TrCP and Endo-Lysosomal Trafficking

The interferon-induced transmembrane protein BST-2/CD317 (tetherin) restricts the release of diverse enveloped viruses from infected cells. The HIV-1 accessory protein Vpu antagonizes this restriction by an unknown mechanism that likely involves the down-regulation of BST-2 from the cell surface. Here, we show that the optimal removal of BST-2 from the plasma membrane by Vpu requires the cellular protein β-TrCP, a substrate adaptor for a multi-subunit SCF E3 ubiquitin ligase complex and a known Vpu-interacting protein. β-TrCP is also required for the optimal enhancement of virion-release by Vpu. Mutations in the DSGxxS β-TrCP binding-motif of Vpu impair both the down-regulation of BST-2 and the enhancement of virion-release. Such mutations also confer dominant-negative activity, consistent with a model in which Vpu links BST-2 to β-TrCP. Optimal down-regulation of BST-2 from the cell surface by Vpu also requires the endocytic clathrin adaptor AP-2, although the rate of endocytosis is not increased; these data suggest that Vpu induces post-endocytic membrane trafficking events whose net effect is the removal of BST-2 from the cell surface. In addition to its marked effect on cell-surface levels, Vpu modestly decreases the total cellular levels of BST-2. The decreases in cell-surface and intracellular BST-2 are inhibited by bafilomycin A1, an inhibitor of endosomal acidification; these data suggest that Vpu induces late endosomal targeting and partial degradation of BST-2 in lysosomes. The Vpu-mediated decrease in surface expression is associated with reduced co-localization of BST-2 and the virion protein Gag along the plasma membrane. Together, the data support a model in which Vpu co-opts the β-TrCP/SCF E3 ubiquitin ligase complex to induce endosomal trafficking events that remove BST-2 from its site of action as a virion-tethering factor.     

Production of membrane proteins using cell–free expression systems

Different overexpression systems are widely used in the laboratory to produce proteins in a reasonable amount for functional and structural studies. However, to optimize these systems without modifying the cellular functions of the living organism remains a challenging task. Cell-free expression systems have become a convenient method for the high-throughput expression of recombinant proteins, and great effort has been focused on generating high yields of proteins. Furthermore, these systems represent an attractive alternative for producing difficult-to-express proteins, such as membrane proteins. In this review, we highlight the recent improvements of these cell-free expression systems and their direct applications in the fields of membrane proteins production, protein therapy and modern proteomics.     

Controlled embryoid body formation via surface modification and avidin–biotin cross-linking

Cell–cell interaction is an integral part of embryoid body (EB) formation controlling 3D aggregation. Manipulation of embryonic stem (ES) cell interactions could provide control over EB formation. Studies have shown a direct relationship between EB formation and ES cell differentiation. We have previously described a cell surface modification and cross-linking method for influencing cell–cell interaction and formation of multicellular constructs. Here we show further characterisation of this engineered aggregation. We demonstrate that engineering accelerates ES cell aggregation, forming larger, denser and more stable EBs than control samples, with no significant decrease in constituent ES cell viability. However, extended culture ≥5 days reveals significant core necrosis creating a layered EB structure. Accelerated aggregation through engineering circumvents this problem as EB formation time is reduced. We conclude that the proposed engineering method influences initial ES cell-ES cell interactions and EB formation. This methodology could be employed to further our understanding of intrinsic EB properties and their effect on ES cell differentiation.     

Autophagy modulates cell migration and β1 integrin membrane recycling

Cell migration is dependent on a series of integrated cellular events including the membrane recycling of the extracellular matrix receptor integrins. In this paper, we investigate the role of autophagy in regulating cell migration. In a wound-healing assay, we observed that autophagy was reduced in cells at the leading edge than in cells located rearward. These differences in autophagy were correlated with the robustness of MTOR activity. The spatial difference in the accumulation of autophagic structures was not detected in rapamycin-treated cells, which had less migration capacity than untreated cells. In contrast, the knockdown of the autophagic protein ATG7 stimulated cell migration of HeLa cells. Accordingly, atg3^?/? and atg5^?/? MEFs have greater cell migration properties than their wild-type counterparts. Stimulation of autophagy increased the co-localization of β1 integrin-containing vesicles with LC3-stained autophagic vacuoles. Moreover, inhibition of autophagy slowed down the lysosomal degradation of internalized β1 integrins and promoted its membrane recycling. From these findings, we conclude that autophagy regulates cell migration, a central mechanism in cell development, angiogenesis, and tumor progression, by mitigating the cell surface expression of β1 integrins.     

Epstein–Barr virus latent membrane protein 1 increases chemo-resistance of cancer cells via cytoplasmic sequestration of Pim-1

Improved treatment of EBV positive lymphoma depends on the identification of molecular mechanism underlying chemo-resistance. LMP1 is an essential transmembrane protein for EBV-induced immortalization of hematopoietic cells. Herein, we show that an oncogenic Pim-1 is translocated to the cytoplasm by LMP1. Three lines of evidence indicate that cytoplasmic sequestration of Pim-1 may be required for LMP1-induced cancer cell survival. First, Pim-1 enhanced the survival of LMP1-overexpressing cells treated with doxorubicin. Second, nuclear export of Pim-1 was sufficient to increase the survival. Third, knockdown of Pim-1 effectively suppressed LMP-1-induced survival of cancer cells. Collectively, these data suggest that Pim-1 is a downstream target of LMP1, and that it contributes to the chemo-resistance of cancer cells.     

Tethering,evagination, and vesiculation via cell–cell interactions in microvascular flow

Biomechanics and Modeling in Mechanobiology - Vesiculation is a ubiquitous process undergone by most cell types and serves a variety of vital cell functions; vesiculation from erythrocytes, in...     

Effects of fatty acid unsaturation numbers on membrane fluidity and α-secretase-dependent amyloid precursor protein processing

Fatty acids may integrate into cell membranes to change physical properties of cell membranes, and subsequently alter cell functions in an unsaturation number-dependent manner. To address the roles of fatty acid unsaturation numbers in cellular pathways of Alzheimer's disease (AD), we systematically investigated the effects of fatty acids on cell membrane fluidity and α-secretase-cleaved soluble amyloid precursor protein (sAPP(α)) secretion in relation to unsaturation numbers using stearic acid (SA, 18:0), oleic acid (OA, 18:1), linoleic acid (LA, 18:2), α-linolenic acid (ALA, 18:3), arachidonic acid (AA, 20:4), eicosapentaenoic acid (EPA, 20:5), and docosahexaenoic acid (DHA, 22:6). Treatments of differentiated human neuroblastoma (SH-SY5Y cells) with AA, EPA and DHA for 24h increased sAPP(α) secretion and membrane fluidity, whereas those treatments with SA, OA, LA and ALA did not. Treatments with AA and DHA did not alter the total expressions of amyloid precursor protein (APP) and α-secretases in SH-SY5Y cells. These results suggested that not all unsaturated fatty acids but only those with 4 or more double bonds, such as AA, EPA and DHA, are able to increase membrane fluidity and lead to increase in sAPP(α) secretion. This study provides insights into dietary strategies for the prevention of AD.     

What is the role of autophagy in HIV-1 infection?

HIV-1 infection is characterized by a progressive CD4 T cell depletion. It is now accepted that apoptosis of uninfected bystander CD4 T lymphocytes plays a major role in AIDS development. Viral envelope glycoproteins (Env) are mainly involved in inducing this cell death process, but the mechanisms triggered by HIV-1 leading to immunodeficiency are still poorly understood. Recently, we have demonstrated that autophagy is a prerequisite for Env-mediated apoptosis in uninfected CD4 T cells, underlining its role in HIV-1 infection. However, occurrence of autophagy in HIV-1-infected cells has not yet been described. Several hypotheses are discussed, based on the comparison with data from other viral infections.     

EGCG inhibited bladder cancer SW780 cell proliferation and migration both in vitro and in vivo via down-regulation of NF-κB and MMP-9

Epigallocatechin-3-gallate (EGCG), the bioactive polyphenol in green tea, has been demonstrated to have various biological activities. Our study aims to investigate the antiproliferation and antimigration effects of EGCG against bladder cancer SW780 cells both in vitro and in vivo. Our results showed that treatment of EGCG resulted in significant inhibition of cell proliferation by induction of apoptosis, without obvious toxicity to normal bladder epithelium SV-HUC-1 cells. EGCG also inhibited SW780 cell migration and invasion at 25–100 μM. Western blot confirmed that EGCG induced apoptosis in SW780 cells by activation of caspases-8, -9 and -3, Bax, Bcl-2 and PARP. Besides, animal study demonstrated that EGCG [100 mg/kg, intraperitoneal (i.p.) injection daily for 3 weeks] decreased the tumor volume significantly in mice bearing SW780 tumors, as well as the tumor weight (decreased by 68.4%). In addition, EGCG down-regulated the expression of nuclear factor-kappa B (NF-κB) and matrix metalloproteinase (MMP)-9 in both protein and mRNA level in tumor and SW780 cells. When NF-κB was inhibited, EGCG showed no obvious effect in cell proliferation and migration. In conclusion, our study demonstrated that EGCG was effective in inhibition SW780 cell proliferation and migration, and presented first evidence that EGCG inhibited SW780 tumor growth by down-regulation of NF-κB and MMP-9.     

What is the role of autophagy in HIV-1 infection?

HIV-1 infection is characterized by a progressive CD4 T cell depletion. It is now accepted that apoptosis of uninfected bystander CD4 T lymphocytes plays a major role in AIDS development. Viral envelope glycoproteins (Env) are mainly involved in inducing this cell death process, but the mechanisms triggered by HIV-1 leading to immunodeficiency are still poorly understood. Recently, we have demonstrated that autophagy is a prerequisite for Env-mediated apoptosis in uninfected CD4 T cells, underlining its role in HIV-1 infection. However, occurrence of autophagy in HIV-1-infected cells has not yet been described. Several hypotheses are discussed, based on the comparison with data from other viral infections.     

Reduction of inflammation and T cell activation after 6 months of cART initiation during acute,but not in early chronic HIV-1 infection

Objectives

To investigate the impact of early combined antiretroviral therapy (cART) on inflammation biomarkers and immune activation during acute and early chronic HIV-1 infection.

Methods

We included 12 acute (AHI), 11 early chronic (EcHI), and 18 late chronic HIV-1-infected (LcHI) individuals who were treated with cART and 18 HIV-1-uninfected (HIV-neg) individuals. Plasmatic levels of inflammation biomarkers, CD8⁺CD38⁺HLA-DR⁺ T cell frequencies, CD4 T cell counts, CD4/CD8 ratio, total HIV-1 DNA and plasmatic viral load were evaluated. Mann–Whitney test, Pearson and Spearman correlation, and linear regression models were used for statistical analyses.

Results

IP-10, IL-18, and sCD163 were significantly elevated at pre-ART in the AHI and EcHI groups, showing a significant reduction after 6 months of cART in the AHI group, achieving similar levels to the HIV-neg group. For the EcHI group, the IP-10 and sCD163 levels were also significantly reduced on M6-ART; however, IP-10 levels remained higher than in the HIV-neg group, and no significant reduction of IL-18 levels was observed. The CD8⁺ T cell activation levels were elevated in the AHI and EcHI groups at pre-ART and showed a significant reduction on M6-ART, but they were similar to levels seen for HIV-neg only after 12 months of cART. At pre-ART, IP-10 levels but not IL-18 levels were positively correlated with HIV-1 viral load in the AHI group.

Conclusions

Early initiation of cART in HIV infection can reduce systemic inflammation, but the earlier normalization of the inflammation markers was only observed when cART was initiated in the acute phase of infection. A slower dynamic of reduction was observed for CD8⁺ T cell activation.