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Objective. To investigate the association of ␤ 2 -glycoprotein I (␤ 2 GPI) with lipid rafts in monocytic cells and to evaluate the proinflammatory and procoagulant effects of anti-␤ 2 GPI binding to its target antigen on the monocyte plasma membrane.Methods. Human monocytes were fractionated by sucrose density-gradient centrifugation and analyzed by Western blotting. Immunoprecipitation experiments were performed to analyze the association of ␤ 2 GPI with lipid rafts and the possible interaction of ␤ 2 GPI with annexin A2 and Toll-like receptor 4 (TLR-4). Monocytes were then stimulated with affinity-purified anti-␤ 2 GPI antibodies from patients with the antiphospholipid syndrome (APS). Interleukin-1 receptor-associated kinase (IRAK) phosphorylation and NF-B activation were evaluated by immunoprecipitation and transcription factor assay, respectively. Supernatants from monocytes were tested for tumor necrosis factor ␣ (TNF␣) and tissue factor (TF) levels by enzyme-linked immunosorbent assay.Results. We found ␤ 2 GPI and its putative receptor annexin A2 in lipid raft fractions of human monocytes. Moreover, there was an association between ␤ 2 GPI and TLR-4, suggesting that it was partially dependent on raft integrity. Triggering with anti-␤ 2 GPI antibodies induced IRAK phosphorylation and consequent NF-B activation, which led to the release of TNF␣ and TF.Conclusion. Anti-␤ 2 GPI antibodies react with their target antigen, likely in association with annexin A2 and TLR-4, in lipid rafts in the monocyte plasma membrane. Anti-␤ 2 GPI binding triggers IRAK phosphorylation and NF-B translocation, leading to a proinflammatory and procoagulant monocyte phenotype characterized by the release of TNF␣ and TF, respectively. These findings provide new insight into the pathogenesis of APS, improving our knowledge of valuable therapeutic targets.

Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltagedependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-b-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.

Mitochondria-associated membranes (MAMs) are subdomains of the endoplasmic reticulum (ER) that interact with mitochondria. This membrane scrambling between ER and mitochondria appears to play a critical role in the earliest steps of autophagy. Recently, lipid microdomains, i.e. lipid rafts, have been identified as further actors of the autophagic process. In the present work, a series of biochemical and molecular analyses has been carried out in human fibroblasts with the specific aim of characterizing lipid rafts in MAMs and to decipher their possible implication in the autophagosome formation. In fact, the presence of lipid microdomains in MAMs has been detected and, in these structures, a molecular interaction of the ganglioside GD3, a paradigmatic "brick" of lipid rafts, with core-initiator proteins of autophagy, such as AMBRA1 and WIPI1, was revealed. This association seems thus to take place in the early phases of autophagic process in which MAMs have been hypothesized to play a key role. The functional activity of GD3 was suggested by the experiments carried out by knocking down ST8SIA1 gene expression, i.e., the synthase that leads to the ganglioside formation. This experimental condition results in fact in the impairment of the ER-mitochondria crosstalk and the subsequent hindering of autophagosome nucleation. We thus hypothesize that MAM raft-like microdomains could be pivotal in the initial organelle scrambling activity that finally leads to the formation of autophagosome.

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