The acridine orange derivative, l0N-nonyl acridine orange, is an appropriate marker of the inner mitochondrial membrane in whole cells. We use membrane model systems to demonstrate that 10N-nonyl acridine orange binds to negatively charged phospholipids (cardiolipin, phosphatidylinositol and phosphatidylserine). The stoichiometry has been found to be 2 mol 10N-nonyl acridine orange/ mol cardiolipin and 1 mol dye/mol phosphatidylscrine or phosphatidylinositol, while, with zwitterionic phospholipids, significant binding could not be detected. The affinity constants were 2 x 1 O6 M ~ for cardiolipin-I ON-nonyl-acridine-orange association and only 7 x lo4 M ~ ' for that of phosphatidylserine and phosphatidylinositol association. The high affinity of the dye for cardiolipin may be explained by two essential interactions; firstly an electrostatic interaction betwcen the quaternary ammonium of nonyl acridine orange and the ionized phosphate residues of cardiolipin and secondly, hydrophobic interactions between adjacent chromophores. A linear relationship was demonstrated between the cardiolipin content of model membranes and the incorporated dye. Consequently, a convenient and rapid method for cardiolipin quantification in membranes was established and applied to the cardiolipin-containing organelle, the mitochondrion.The 10N-nonyl acridine orange (NAO), which is specifically incorporated into the inner mitochondrial membrane [l], plays a prominent role in the study of mitochondria in whole cells [2, 31. It enables monitoring of mitochondria in different situations, such as the cell cycle [4] and cell ageing [5] and also discrimination between different subpopulations of a heterogenous cell population, according to thcir mitochondrial contents [6, 71. Nevertheless, up to now the membrane molccular species which are specifically recognized by NAO, have not been determined.The large number of inner-mitochondrial-membrane enzymes implicated in oxidative phosphorylation, differing in their conformation and biological properties require a similar lipid environment for their activity. Cardiolipin, one of the three major phospholipids present in the inner membrane [8 -121, has been reported to be essential for the activity of the ADP/ATP carrier protein [I 31, for the phosphate transport protein [14] and for various other enzyme complexes [15,16]. This phospholipid has also been reported to be associated with the F1-FO ATPase [17]. Consequently, the NAO inhibition of the inner-mitochondrial-membrane enzymes [ 181 may be due to the interaction between the positively charged dye and cardiolipin, the main acidic phospholipid present in the inner mitochondrial membrane.The purpose ofthis work was to establish, with reference to different model membranes, that lON-nonyl acridine orange intcracts with acidic phospholipids and, more particularly, with cardiolipin. The absorbance spectra of NAO incubated with liposomes and measurements of the degree of saturation allowed us to determine the specificity and the stoichiometry of NAO ...

Glioblastoma multiforme is the most common and the most aggressive primary brain tumor. It is characterized by a high degree of hypoxia and also by a remarkable resistance to therapy because of its adaptation capabilities that include autophagy. This degradation process allows the recycling of cellular components, leading to the formation of metabolic precursors and production of adenosine triphosphate. Hypoxia can induce autophagy through the activation of several autophagy-related proteins such as BNIP3, AMPK, REDD1, PML, and the unfolded protein response-related transcription factors ATF4 and CHOP. This review summarizes the most recent data about induction of autophagy under hypoxic condition and the role of autophagy in glioblastoma.

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The distribution of cardiolipin across the inner mitochondrial membrane was directly determined by using the ability of the fluorescent dye 1 O-N-nonyl-3,6-bis(dimethylamino)acridine (10-N-nonyl acridine orange) to form dimers when it interacts with the diacidic phospholipid. Two independent methods were employed : (a) a spectrophotometric measurement of 10-N-nonyl acridine orange binding to isolated rat liver mitochondria, mitoplasts and inside-out submitochondrial particles, and (b) a flow-cytometric analysis of specific red fluorescence, emitted when two dye molecules are bound to one membrane cardiolipin ; the stoichiometry of 10-N-nonyl acridine orange binding to phosphatidylserine and phosphatidylinositol, 1 mol dye/mol phospholipid, prevented dye dimerisation and subsequent red-fluorescence appearance. 57% total cardiolipin was present in the outer leaflets of inner membranes of isolated organelles, a distribution confirmed by saturation measurements for mitoplasts and inside-out submitochondrial particles. The same asymmetry was directly observed in situ with mitochondrial membranes of quiescent L1210 cells, and with mitochondrial membranes of respiring yeasts. Nevertheless, alterations in ATP synthesis and inhibition of mitochondrial protein synthesis revealed that cardiolipin distribution was apparently tightly correlated with mitochondrial membrane assembly and activity.

Studies have established that autophagy constitutes an efficient process to recycle cellular components and certain proteins. The phenomenon was demonstrated primarily in response to nutrient starvation, and there are increasing evidences that it is implied in differentiation. Keratinocyte differentiation was going along an activation of lysosomal enzymes and organelle clearance, and terminal steps are sometimes described as a specialized form of cell death leading to corneocytes. We examined whether initiation of the process in human keratinocyte HaCaT involves autophagy. The KSFM™ culture medium was substituted by M199, which contains a low glucose concentration but a high calcium level (known to induce differentiation). Metabolic stress reduced enhanced cell number in G(1) phase, without apoptotic features (ΔΨmt and membrane integrity are unchanged). Morphological changes were associated with a lower integrin ß1 expression and modifications of protein levels involved in keratinocyte differentiation (involucrin, keratin K10 and ΔNp63α). Whereas autophagic signalling was supported by SIRT1 and pAMPK (T172) increase according to time kinetic, which led to the disappearance of mTOR phosphorylated on S2448 residue. The significant Bcl-X(L) level reduction with stress promoted autophagy, by the release of Beclin-1, whereas ATG5-ATG12 and LC3-II that are involved in autophagosome formation were enhanced significantly. Then, the level of lysosomal protein cathepsin B rose to execute autophagy. Kinetic studies established that autophagy would constitute an early signalling process required for keratinocyte commitment in differentiation pathway.