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 ...

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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.

Transmembrane asymmetry of cardiolipin in yeast was monitored during the switch from fermentative to gluconeogenic growth and the reverse. As soon as cells used ethanol as an electron donor to produce ATP by oxidative phosphorylation, rapid and abundant cardiolipin synthesis was observed on the matrix side of the inner mitochondrial membrane followed by a transverse rearrangement between the two leaflets. The cardiolipin distribution changed from about 20:80 (in/out) to 70:30 (in/out), and after translocation towards the outer leaflet it finally became 37:63 (in/out). At the same time, cytochrome c oxidase activity remained stable, then increased as a possible result of the topographical rearrangement. During the reverse process from gluconeogenic to fermentative growth, the amount of cardiolipin rapidly decreased by half, its bilayer distribution apparently changing to a monolayer organization before the 20:80 (in/out) asymmetry of repressed cells was re-established. Experimental impairment of cardiolipin topography by antibiotic inhibition of gene expression or in situ dissipation of mitochondrial membrane potential produced data that prove that the amount and transmembrane distribution of the phospholipid are two specific parameters of the mitochondrial inner membrane organization in both fermentative (2.2 fmol/cell and 20:80, in/out) and gluconeogenic (4.2 fmol/cell and 37:63, in/out) growing yeast cells. Finally, the inner mitochondrial membrane topography of cardiolipin appeared to be closely associated with the transmembrane redox potential.

Due to its spectral characteristics, the fluorochrome nonyl acridine orange (NAO) (&,,:489 nm, X,,:525 nm), which is spontaneously incorporated by mitochondria with a high relative specificity, provides a new probe for the in situ study of these organelles by flow cytometry. In 15 min at 20°C, the dye at 4.75 x M saturates the mitochondrial binding sites present in 1.5 x lo6 cells. Unlike Rh 123, the fixation of the probe is not affected by the action of uncouplers and ionophores. Unlike acridine orange, its binding is not sensitive to nucleases.By studying the mitochondrial incorporation of the fluorochrome during the cell cycle of murine splenocytes, it was possible to show that the biogenesis of NAO-stained mitochondrial constituents mainly occurs during the GI phase.Key terms: Acridines, mitochondria analysis, fluorescent probes, cell cycle, flow cytometry For the past 10 years, the in situ study of mitochondria has been carried out mainly through the use of a mitochondria-specific fluorochrome, rhodamine 123 (Rh 123). Initially, Johnson et al. (17)showed by microscopy that this dye directly and specifically stains the mitochondrial membranes and that its incorporation depends on the mitochondrial transmembrane potential (18).Coupled with analysis by flow cytometry (FCM), probe incorporation has made possible quantitative studies of the mitochondrial compartment (5). Applications were extended to the study of mitochondria in normal and transformed cells (16,21) during proliferation (2,9,10, 15,23,24,26) or differentiation (6,301. Furthermore, rhodamine 123 was used to monitor the effects of various drugs on the transmembrane potential (1,3,14,19). Although these investigations have improved our understanding of the interactions between the nuclear and mitochondrial cell compartments, in particular during division, it was found that fluorochrome incorporation may indicate both a change in the activity of the organelle and a change in its true membrane mass. In order to carry out in situ studies, it was therefore necessary to obtain fluorochromes that bind to mitochondria according to a mechanism independent of respiratory activity.Such a dye would make it possible to determine more accurately than with rhodamine 123 the total mitochondrial mass or the mass of one or several of its constituents, and, in some cases, the size and number of organelles.Among the numerous mitochondria-specific fluorochromes described recently (22,25), it has been shown that 10-nonyl acridine orange (NAO), synthesized by the team of Professor H.W. Zimmermann (12,13,28,29), binds to these organelles in a manner independent of the respiratory activity. Moreover, the spectral properties of NAO allow an optimum excitation at approximately 488 nm, and the fluorescence may be detected between 500 and 600 nm with a n emission maximum at 525 nm.In this study we therefore chose to determine the conditions for use of this fluorochrome in quantitative FCM analysis. NAO is rapidly and spontaneously incorporated by murine splenocytes following c...