Role of mitochondrial raft-like microdomains in the regulation of cell apoptosis

Lipid rafts are envisaged as lateral assemblies of specific lipids and proteins that dissociate and associate rapidly and form functional clusters in cell membranes. These structural platforms are not confined to the plasma membrane; indeed lipid microdomains are similarly formed at subcellular organelles, which include endoplasmic reticulum, Golgi and mitochondria, named raft-like microdomains. In addition, some components of raft-like microdomains are present within ER-mitochondria associated membranes. This review is focused on the role of mitochondrial raft-like microdomains in the regulation of cell apoptosis, since these microdomains may represent preferential sites where key reactions take place, regulating mitochondria hyperpolarization, fission-associated changes, megapore formation and release of apoptogenic factors. These structural platforms appear to modulate cytoplasmic pathways switching cell fate towards cell survival or death. Main insights on this issue derive from some pathological conditions in which alterations of microdomains structure or function can lead to severe alterations of cell activity and life span. In the light of the role played by raft-like microdomains to integrate apoptotic signals and in regulating mitochondrial dynamics, it is conceivable that these membrane structures may play a role in the mitochondrial alterations observed in some of the most common human neurodegenerative diseases, such as Amyotrophic lateral sclerosis, Huntington's chorea and prion-related diseases. These findings introduce an additional task for identifying new molecular target(s) of pharmacological agents in these pathologies

Requirements for Membrane Attack Complex Formation and Anaphylatoxins Binding to Collagen-Activated Platelets

The activation of complement during platelet activation is incompletely understood.We sought to explore the formation of C5b-9 and anaphylatoxins binding to collagen-activated platelets.C5b-9, anaphylatoxins C3a, C4a and C5a, and anaphylatoxin receptors C3aR1 and C5aR were measured by flow cytometry and/or confocal microscopy. Platelet microparticles were quantified by flow cytometry, and their C5b-9 content was determined by western blot analyses. In all experiments, sodium citrate was used for blood anticoagulation.C5b-9 rapidly formed on the platelet surface following activation with collagen, TRAP, ADP or A23187, but was surprisingly restricted to a subset of platelets (1 to 15%) independently of P-selectin or phosphatidylserine exposure. Following collagen activation, C5b-9-positive platelets in thrombi were found associated with collagen fibres. C5b-9 formation was obliterated by Mg(2+)-EGTA and significantly reduced by the thrombin inhibitor hirudin (-37%, p<0.05), but was unaffected by chondroitinase, compstatin, SCH79797 (PAR-1 inhibitor), or in the PRP of a MBL-deficient donor. Compstatin and Mg(2+)-EGTA, but not hirudin, SCH79797 or chondroitinase, inhibited the formation of collagen-induced microparticles (-71% and -44%, respectively, p<0.04). These microparticles contained greater amounts of C5b-9 compared with the other agonists. Platelet activation by collagen or convulxin resulted in the strong binding of anaphylatoxins and the exposure of receptors C3aR1 and C5aR (CD88) on their surface.C5b-9 formation on collagen-activated platelets is i) partially controlled by thrombin, ii) restricted to a subset of platelets, and iii) can occur without P-selectin expression or phosphatidylserine exposure. Activated platelets bind anaphylatoxins on their surface and express C3a and C5a receptors, which may contribute to the localization of inflammatory processes during thrombosis

Role of GD3-CLIPR-59 Association in Lymphoblastoid T Cell Apoptosis Triggered by CD95/Fas

We previously found that a directional movement of the raft component GD3 towards mitochondria, by its association with microtubules, was mandatory to late apoptogenic events triggered by CD95/Fas. Since CLIPR-59, CLIP-170-related protein, has recently been identified as a microtubule binding protein associated with lipid rafts, we analyzed the role of GD3-CLIPR-59 association in lymphoblastoid T cell apoptosis triggered by CD95/Fas. To test whether CLIPR-59 could play a role at the raft-microtubule junction, we performed a series of experiments by using immunoelectron microscopy, static or flow cytometry and biochemical analyses. We first assessed the presence of CLIPR-59 molecule in lymphoblastoid T cells (CEM). Then, we demonstrated that GD3-microtubule interaction occurs via CLIPR-59 and takes place at early time points after CD95/Fas ligation, preceding the association GD3-tubulin. GD3-CLIPR-59 association was demonstrated by fluorescence resonance energy transfer (FRET) analysis. The key role of CLIPR-59 in this dynamic process was clarified by the observation that silencing CLIPR-59 by siRNA affected the kinetics of GD3-tubulin association, spreading of GD3 towards mitochondria and apoptosis execution. We find that CLIPR-59 may act as a typical chaperone, allowing a prompt interaction between tubulin and the raft component GD3 during cell apoptosis triggered by CD95/Fas. On the basis of the suggested role of lipid rafts in conveying pro-apoptotic signals these results disclose new perspectives in the understanding of the mechanisms by which raft-mediated pro-apoptotic signals can directionally reach their target, i.e. the mitochondria, and trigger apoptosis execution

Temporal resolution of protein–protein interactions in the live-cell plasma membrane

We have recently devised a method to quantify interactions between a membrane protein (“bait”) and a fluorophore-labeled protein (“prey”) directly in the live-cell plasma membrane (Schwarzenbacher et al. Nature Methods 5:1053–1060 2008). The idea is to seed cells on surfaces containing micro-patterned antibodies against the exoplasmic domain of the bait, and monitor the co-patterning of the fluorescent prey via fluorescence microscopy. Here, we characterized the time course of bait and prey micropattern formation upon seeding the cells onto the micro-biochip. Patterns were formed immediately after contact of the cells with the surface. Cells were able to migrate over the chip surface without affecting the micropattern contrast, which remained constant over hours. On single cells, bait contrast may be subject to fluctuations, indicating that the bait can be released from and recaptured on the micropatterns. We conclude that interaction studies can be performed at any time-point ranging from 5 min to several hours post seeding. Monitoring interactions with time opens up the possibility for new assays, which are briefly sketched in the discussion section

Syndecans Reside in Sphingomyelin-Enriched Low-Density Fractions of the Plasma Membrane Isolated from a Parathyroid Cell Line

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are one of the basic constituents of plasma membranes. Specific molecular interactions between HSPGs and a number of extracellular ligands have been reported. Mechanisms involved in controlling the localization and abundance of HSPG on specific domains on the cell surface, such as membrane rafts, could play important regulatory roles in signal transduction. METHODOLOGY/PRINCIPAL FINDINGS: Using metabolic radiolabeling and sucrose-density gradient ultracentrifugation techniques, we identified [(35)S]sulfate-labeled macromolecules associated with detergent-resistant membranes (DRMs) isolated from a rat parathyroid cell line. DRM fractions showed high specific radioactivity ([(35)S]sulfate/mg protein), implying the specific recruitment of HSPGs to the membrane rafts. Identity of DRM-associated [(35)S]sulfate-labeled molecules as HSPGs was confirmed by Western blotting with antibodies that recognize heparan sulfate (HS)-derived epitope. Analyses of core proteins by SDS-PAGE revealed bands with an apparent MW of syndecan-4 (30-33 kDa) and syndecan-1 (70 kDa) suggesting the presence of rafts with various HSPG species. DRM fractions enriched with HSPGs were characterized by high sphingomyelin content and found to only partially overlap with the fractions enriched in ganglioside GM1. HSPGs could be also detected in DRMs even after prior treatment of cells with heparitinase. CONCLUSIONS/SIGNIFICANCE: Both syndecan-1 and syndecan-4 have been found to specifically associate with membrane rafts and their association seemed independent of intact HS chains. Membrane rafts in which HSPGs reside were also enriched with sphingomyelin, suggesting their possible involvement in FGF signaling. Further studies, involving proteomic characterization of membrane domains containing HSPGs might improve our knowledge on the nature of HSPG-ligand interactions and their role in different signaling platforms

An intranasal selective antisense oligonucleotide impairs lung cyclooxygenase-2 production and improves inflammation, but worsens airway function, in house dust mite sensitive mice

<p>Abstract</p> <p>Background</p> <p>Despite its reported pro-inflammatory activity, cyclooxygenase (COX)-2 has been proposed to play a protective role in asthma. Accordingly, COX-2 might be down-regulated in the airway cells of asthmatics. This, together with results of experiments to assess the impact of COX-2 blockade in ovalbumin (OVA)-sensitized mice in vivo, led us to propose a novel experimental approach using house dust mite (HDM)-sensitized mice in which we mimicked altered regulation of COX-2.</p> <p>Methods</p> <p>Allergic inflammation was induced in BALBc mice by intranasal exposure to HDM for 10 consecutive days. This model reproduces spontaneous exposure to aeroallergens by asthmatic patients. In order to impair, but not fully block, COX-2 production in the airways, some of the animals received an intranasal antisense oligonucleotide. Lung COX-2 expression and activity were measured along with bronchovascular inflammation, airway reactivity, and prostaglandin production.</p> <p>Results</p> <p>We observed impaired COX-2 mRNA and protein expression in the lung tissue of selective oligonucleotide-treated sensitized mice. This was accompanied by diminished production of mPGE synthase and PGE₂in the airways. In sensitized mice, the oligonucleotide induced increased airway hyperreactivity (AHR) to methacholine, but a substantially reduced bronchovascular inflammation. Finally, mRNA levels of hPGD synthase remained unchanged.</p> <p>Conclusion</p> <p>Intranasal antisense therapy against COX-2 in vivo mimicked the reported impairment of COX-2 regulation in the airway cells of asthmatic patients. This strategy revealed an unexpected novel dual effect: inflammation was improved but AHR worsened. This approach will provide insights into the differential regulation of inflammation and lung function in asthma, and will help identify pharmacological targets within the COX-2/PG system.</p

Sperm protein 17 is expressed in human nervous system tumours

BACKGROUND: Human sperm protein 17 (Sp17) is a highly conserved protein that was originally isolated from a rabbit epididymal sperm membrane and testis membrane pellet. It has recently been included in the cancer/testis (CT) antigen family, and shown to be expressed in multiple myeloma and ovarian cancer. We investigated its immunolocalisation in specimens of nervous system (NS) malignancies, in order to establish its usefulness as a target for tumour-vaccine strategies. METHODS: The expression of Sp17 was assessed by means of a standardised immunohistochemical procedure [(mAb/antigen) MF1/Sp17] in formalin-fixed and paraffin embedded surgical specimens of NS malignancies, including 28 neuroectodermal primary tumours (6 astrocytomas, 16 glioblastoma multiforme, 5 oligodendrogliomas, and 1 ependymoma), 25 meningeal tumours, and five peripheral nerve sheath tumours (4 schwannomas, and 1 neurofibroma),. RESULTS: A number of neuroectodermal (21%) and meningeal tumours (4%) were found heterogeneously immunopositive for Sp17. None of the peripheral nerve sheath tumours was immunopositive for Sp17. The expression pattern was heterogeneous in all of the positive samples, and did not correlate with the degree of malignancy. CONCLUSION: The frequency of expression and non-uniform cell distribution of Sp17 suggest that it cannot be used as a unique immunotherapeutic target in NS cancer. However, our results do show the immunolocalisation of Sp17 in a proportion of NS tumour cells, but not in their non-pathological counterparts. The emerging complex function of Sp17 makes further studies necessary to clarify the link between it and immunopositive cells

Dynamics of HIV-1 Assembly and Release

Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1–2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of ∼5×10−3 s−1, corresponding to 8–9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at ∼1,500±700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics

Release of Intracellular Calcium Stores Facilitates Coxsackievirus Entry into Polarized Endothelial Cells

Group B coxsackieviruses (CVB) are associated with viral-induced heart disease and are among the leading causes of aseptic meningitis worldwide. Here we show that CVB entry into polarized brain microvasculature and aortic endothelial cells triggers a depletion of intracellular calcium stores initiated through viral attachment to the apical attachment factor decay-accelerating factor. Calcium release was dependent upon a signaling cascade that required the activity of the Src family of tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3. CVB-mediated calcium release was required for the activation of calpain-2, a calcium-dependent cysteine protease, which controlled the vesicular trafficking of internalized CVB particles. These data point to a specific role for calcium signaling in CVB entry into polarized endothelial monolayers and highlight the unique signaling mechanisms used by these viruses to cross endothelial barriers

Lack of Chemokine Signaling through CXCR5 Causes Increased Mortality, Ventricular Dilatation and Deranged Matrix during Cardiac Pressure Overload

RATIONALE: Inflammatory mechanisms have been suggested to play a role in the development of heart failure (HF), but a role for chemokines is largely unknown. Based on their role in inflammation and matrix remodeling in other tissues, we hypothesized that CXCL13 and CXCR5 could be involved in cardiac remodeling during HF. OBJECTIVE: We sought to analyze the role of the chemokine CXCL13 and its receptor CXCR5 in cardiac pathophysiology leading to HF. METHODS AND RESULTS: Mice harboring a systemic knockout of the CXCR5 (CXCR5(-/-)) displayed increased mortality during a follow-up of 80 days after aortic banding (AB). Following three weeks of AB, CXCR5(-/-) developed significant left ventricular (LV) dilatation compared to wild type (WT) mice. Microarray analysis revealed altered expression of several small leucine-rich proteoglycans (SLRPs) that bind to collagen and modulate fibril assembly. Protein levels of fibromodulin, decorin and lumican (all SLRPs) were significantly reduced in AB CXCR5(-/-) compared to AB WT mice. Electron microscopy revealed loosely packed extracellular matrix with individual collagen fibers and small networks of proteoglycans in AB CXCR5(-/-) mice. Addition of CXCL13 to cultured cardiac fibroblasts enhanced the expression of SLRPs. In patients with HF, we observed increased myocardial levels of CXCR5 and SLRPs, which was reversed following LV assist device treatment. CONCLUSIONS: Lack of CXCR5 leads to LV dilatation and increased mortality during pressure overload, possibly via lack of an increase in SLRPs. This study demonstrates a critical role of the chemokine CXCL13 and CXCR5 in survival and maintaining of cardiac structure upon pressure overload, by regulating proteoglycans essential for correct collagen assembly