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.

No abstract

In this study we analyzed the interaction of prion protein PrP C with components of glycosphingolipid-enriched microdomains in lymphoblastoid T cells. PrP C was distributed in small clusters on the plasma membrane, as revealed by immunoelectron microscopy. PrP C is present in microdomains, since it coimmunoprecipitates with GM3 and the raft marker GM1. A strict association between PrP C and Fyn was revealed by scanning confocal microscopy and coimmunoprecipitation experiments. The phosphorylation protein ZAP-70 was immunoprecipitated by anti-PrP after T cell activation. These results demonstrate that PrP C interacts with ZAP-70, suggesting that PrP C is a component of the multimolecular signaling complex within microdomains involved in T cell activation.

a b s t r a c tCardiolipin (CL) has recently been shown to provide an anchor and an essential activating platform for caspase-8 on mitochondria. We hypothesize that these platforms may correspond to ''raft-like" microdomains, which have demonstrated to be detectable on mitochondrial membrane of cells undergoing apoptosis. The role for CL in ''raft-like" microdomains could be to anchor caspase-8 at contact sites between inner and outer membranes, facilitating its self-activation, Bid cleavage and apoptosis execution. The role played by ''raft-like" microdomains in the apoptotic program could introduce a new task in the pathogenetic studies on human diseases associated with cardiolipin dismetabolism.

It was shown that receptor-mediated apoptosis involves a cascade of subcellular events including alterations of mitochondria. Loss of mitochondrial membrane potential that follows death receptor ligation allows the release of apoptogenic factors that result in apoptosis execution. Further important mitochondrial changes have been observed in this regard: mitochondrial remodeling and fission that appear as prerequisites for the occurrence of the cell death program. As it was observed that lipid rafts, glycosphingolipid-enriched structures, can participate in the apoptotic cascade being recruited to the mitochondria under receptor-mediated proapoptotic stimulation, we decided to analyze the possible implication of these microdomains in mitochondrial fission. We found that molecules involved in mitochondrial fission processes are associated with these domains. In particular, although hFis1 was constitutively included in mitochondrial raft-like domains, dynamin-like protein 1 was recruited to these domains on CD95/Fas triggering. Accordingly, the disruption of rafts, for example, by inhibiting ceramide synthase, leads to the impairment of fission molecule recruitment to the mitochondria, reduction of mitochondrial fission and a significant reduction of apoptosis. We hypothesize that under apoptotic stimulation the recruitment of fission-associated molecules to the mitochondrial rafts could have a role in the morphogenetic changes leading to organelle fission. Maintenance of shape and morphology is required for normal mitochondrial and cellular functions. Mitochondria frequently undergo fission and fusion processes that regulate their morphology, number and function.1,2 This remodeling is of great relevance in both cell life and death being a mitochondrial network involved in all the main cell activities, including proliferation, differentiation and senescence, as well as cell death by apoptosis. In particular, although mitochondrial fusion processes seem to be associated with cell differentiation and senescence, 3 fission processes are mandatory during cell proliferation and, as recently stated, in apoptotic events. Although mitochondrial fusion serves to maintain a tubular mitochondrial network and optimal mitochondrial function, mitochondrial fission is required in dividing cells to ensure inheritance of mitochondria by daughter cells, but it is also important during differentiation in response to new energy demands. 4 Mitochondrial fission and fusion depend on the balance among several fusion and fission proteins. These include mitofusin 1 and 2 (Mfn1, Mfn2) and OPA1, which are large GTPases essential for fusion processes and localized in the mitochondria, associated with the outer (Mfn1 and 2) or inner (OPA1) membrane. [5][6][7] On the fission side, other proteins are involved: dynamin-related protein 1, also known as dynamin-like protein 1 (DLP1), which appears to be recruited to mitochondria to mediate fission activities, 8,9 and the human homologue of mitochondrial fission protein previously found in yeast, t...

These findings support the view that the processing of proteins in autophagy generates citrullinated peptides recognized by the immune system in RA.

Mitochondria-associated membranes (MAMs) are essential communication subdomains of the endoplasmic reticulum (ER) that interact with mitochondria. We previously demonstrated that, upon macroautophagy/autophagy induction, AMBRA1 is recruited to the BECN1 complex and relocalizes to MAMs, where it regulates autophagy by interacting with raft-like components. ERLIN1 is an endoplasmic reticulum lipid raft protein of the prohibitin family. However, little is known about its association with the MAM interface and its involvement in autophagic initiation. In this study, we investigated ERLIN1 association with MAM raft-like microdomains and its interaction with AMBRA1 in the regulation of the autophagic process. We show that ERLIN1 interacts with AMBRA1 at MAM raft-like microdomains, which represents an essential condition for autophagosome formation upon nutrient starvation, as demonstrated by knocking down ERLIN1 gene expression. Moreover, this interaction depends on the "integrity" of key molecules, such as ganglioside GD3 and MFN2. Indeed, knocking down ST8SIA1/GD3-synthase or MFN2 expression impairs AMBRA1-ERLIN1 interaction at the MAM level and hinders autophagy. In conclusion, AMBRA1-ERLIN1 interaction within MAM raft-like microdomains appears to be pivotal in promoting the formation of autophagosomes. Abbreviations: ACSL4/ACS4: acyl-CoA synthetase long chain family member 4; ACTB/β-actin: actin beta;