Compared flow cytometric analysis of mitochondria using 10-n-nonyl acridine orange and rhodamine 123

The use of the supravital mitochondrial-specific dye Rhodamine 123 (Rh 123) in combination with flow cytometry permits the monitoring of the changes in the mitochondrial transmembrane potential, reflecting the overall mitochondrial activity of the living cell. While this probe appears to be a potent tool for these studies, it also exhibits an important limit in the interpretation of the results: it cannot distinguish between an increase in mitochondrial activity without biogenesis and a modification of mitochondrial content. 10-n-Nonyl Acridine Orange chloride (NAO) constitutes another mitochondrial specific fluorochrome. In contrast with Rh 123, NAO accumulation in the cell does not seem to be driven by the proton-motrice force but does seem to be related to specific interactions with mitochondrial membrane proteins and/or lipids. In this work, the cytotoxicity of NAO, the kinetics of cellular uptake and the release of the dye have been determined using flow cytometry. The use of several ionophores or mitochondrial inhibitors has confirmed the independence of NAO uptake regarding mitochondrial transmembrane potential. NAO was also used to examine the changes in the mitochondrial compartment during the transfer of articular chondrocytes from cartilage to the culture conditions, where Rh 123 evidenced changes in mitochondrial activity and/or biogenesis, in order to know whether the use of probes with different specificity allows one to distinguish between mitochondrial activity and biogenesis.     

Visualization of phospholipid domains in Escherichia coli by using the cardiolipin-specific fluorescent dye 10-N-nonyl acridine orange

Cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) was used to visualize CL distribution in Escherichia coli cells of different phospholipid compositions. In a filamentous mutant containing only anionic phospholipids, green fluorescent spots were observed along the filaments at approximately regular intervals. Three-dimensional image reconstruction obtained by optical sectioning and a deconvolution algorithm revealed NAO-binding domains in the plane of the cell membrane. Substantial red fluorescence emission of bound NAO supported labeling of CL-containing domains. These structures were not found in mutants deficient in CL biosynthesis. The domains were also observed mostly in the septal region and on the poles in cells of normal size with wild-type phospholipid composition.     

Use of the fluorescent dye 10-N-nonyl acridine orange in quantitative and location assays of cardiolipin: a study on different experimental models

The fluorescent dye 10-N-nonyl acridine orange (NAO) is extensively used for location and quantitative assays of cardiolipin in living cells on the assumption of its high specificity for cardiolipin; however, the limits and the mechanism of this specificity are not clear. Moreover, whether factors such as the membrane potential in mitochondria may limit the consistency of the results obtained by this method is open to discussion. The aim of this research was to investigate the effects of some experimental factors on the selective fluorescence of NAO in the presence of cardiolipin in artificial and natural membranes (mitochondria). The results show that the fluorescence of NAO, due to interaction with cardiolipin, is significantly modified by factors that control the spatial arrangement of cardiolipin molecules within the space of the membrane under investigation. Moreover, the present observations suggest that the specific effect of cardiolipin is to facilitate the dimerization of this fluorescent dye, thus confirming that reliable measurements of cardiolipin concentration can be obtained only when the NAO/cardiolipin molar ratio is equal to 2. The finding is also reported that in isolated respiring mitochondria the interaction of NAO with cardiolipin is somewhat related to the respiratory state of mitochondria.     

Hydrophobic acridine dyes for fluorescence staining of mitochondria in living cells. 1.Thermodynamic and spectroscopic properties of 10-n-alkylacridine orange chlorides

10-n-Alkyl-acridine-orange-chlorides (alkyl-AOs) are excellent dyes for fluorescence staining of mitochondria in living cells. The thermodynamic and spectroscopic properties of the series alkyl = methyl to nonyl have been investigated. The dyes form dimers in aqueous solution. The dimerisation is mainly a consequence of the hydrophobic interaction. The dissociation constant K respectively association constant K-1 of the dimers describes the hydrophobic interaction and therefore the hydrophobic properties of the dye cations. The dissociation constant K = K0 at the standard temperature T = 298 K has been determined spectroscopically in aqueous solution. It depends on the length of the alkyl residue n-CmH2m + 1 (m = 1 - 9) (Table 2). In addition the standard dissociation enthalpies (energies) delta H0 and dissociation entropies delta S0 have been determined from the temperature dependence of K (Table 2). With increasing chain length m the thermodynamic parameters K0, delta H0, delta S0 decrease. Therefore with growing m the dimers are stabilized. This stabilization is an entropic effect which is diminished by the energetic effect. The change of the thermodynamic parameters with m is in agreement with the concept of hydrophobic interaction and the stabilization of water structure in the surroundings of hydrophobic residues. As one would expect nonyl-AO is the most hydrophobic dye of the series. As an example the spectroscopic properties of nonyl-AO have been determined. We measured the absorption, luminescence and polarization spectra in rigid ethanol at 77 K. Under these conditions alkyl-AOs associate like dyes in Water at room temperature. The spectra depend on the concentration of the solution. In very dilute solution we observe mainly the spectra of the monomers M, in concentrated solution the spectra of the dimers D. The spectra of M and D are characteristically different. The monomers have one long wave length absorption M1 = 20.000 cm-1 with resonance fluorescence. In addition there is a long living phosphorescence at 16.600 cm-1. Its polarization is nearly perpendicular to the plane of the AO residue. The dimers have two long wave length absorption bands D1 = 18.700 and D2 = 21.200 cm-1 with very different intensities. D1 has very low intensity and is forbitten, D2 is allowed. D1 shows fluorescence. Phosphorescence has not been observed. D1, D2 and also M1 are polarized in the plane of the AO residue. At short wave length absorption and polarization spectra are very similar. From the spectra we constructed the energy level diagram of M and D (Fig. 9). The first excited state of M splits in D in two levels. The level splitting and the transition i     

Titration of cardiolipin by either 10-<Emphasis Type="Italic">N</Emphasis>-nonyl acridine orange or acridine orange sensitizes the adenine nucleotide carrier to permeability transition

Under the action of carboxyatractyloside or fatty acids, adenine nucleotide translocase switches its function from nucleotide carrier to modulator of the opening of a non-specific pore. In addition to the effect of these agents, this modification in activity is, in some way, dependent on the influence of the lipid milieu of the membrane. Cardiolipin is, among other membrane phospholipids, the one that interacts the most with the translocase. This work shows that 10-N-nonyl acridine orange and acridine orange, probes for this phospholipid, modify the sensitivity of the translocase to carboxyatractyloside, oleate, and palmitate to induce permeability transition. The results also show that these probes stimulate the release of mitochondrial cytochrome c, and increase labeling of the carrier by eosin 5-maleimide. Based on the aforementioned it is proposed that the increase in sensitivity is due to a conformational change in the translocase, induced by the binding of the probe to cardiolipin.     

Targeting of mitochondria by 10-N-alkyl acridine orange analogues: role of alkyl chain length in determining cellular uptake and localization

10-N-Nonyl acridine orange (NAO) is used as a mitochondrial probe because of its high affinity for cardiolipin (CL). Targeting of NAO may also depend on mitochondrial membrane potential. As the nonyl group has been considered essential for targeting, a systematic study of alkyl chain length was undertaken; three analogues (10-methyl-, 10-hexyl-, and 10-hexadecyl-acridine orange) were synthesized and their properties studied in phospholipid monolayers and breast cancer cells. The shortest and longest alkyl chains reduced targeting, whereas the hexyl group was superior to the nonyl group, allowing very clear and specific targeting to mitochondria at concentrations of 20-100 nM, where no evidence of toxicity was apparent. Additional studies in wild-type and cardiolipin-deficient yeast cells suggested that cellular binding was not absolutely dependent upon cardiolipin.     

Variation in human acrocentric chromosomes with acridine orange reverse banding.

Twenty-five normal subjects were studied by acridine orange reverse (RFA) banding in order to obtain a preliminary estimate of the type and frequency of variations in color and length. Color variations were classified into 1 of 6 colors and size variations into 1 of 5 levels. The same cells were also studied by Q banding. Acridine orange reverse banding was found to be more useful than Q banding for characterizing variations in chromosomes 14, 15, 21 and 22. In addition, it was found that there was no consistent relationship between pale or bright Q banding and the various colors observed with RFA banding. For the optimal characterization of a chromosomal variation, multiple banding technics, including RFA banding, are necessary.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria. II. Preferential interaction of cardiolipin with specific molecular species of phospholipid

A specific effect of cardiolipin on fluidity of mitochondrial membranes was demonstrated in Tetrahymena cells acclimated to a lower temperature in the previous report (Yamauchi, T., Ohki, K., Maruyama, H. and Nozawa, Y. (1981) Biochim. Biophys. Acta 649, 385-392). This study was further confirmed by the experiment using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Anisotropy of DPH for microsomal and pellicular total lipids from Tetrahymena cells showed that membrane fluidity of these lipids increased gradually as the cells were incubated at 15 degrees C after the shift down of growth temperature from 39 degrees C. However, membrane fluidity of mitochondrial total lipids was kept constant up to 10 h. This finding is compatible with the result obtained using spin probe in the previous report. Additionally, the break-point temperature of DPH anisotropy was not changed in mitochondrial lipids whereas those temperatures in pellicular and microsomal lipids lowered during the incubation at 15 degrees C. Interaction between cardiolipins and various phospholipids, which were isolated from Tetrahymena cells grown at 39 degrees C or 15 degrees C and synthesized chemically, was investigated extensively using a spin labeling technique. The addition of cardiolipins from Tetrahymena cells grown at either 39 degrees C or 15 degrees C did not change the membrane fluidity (measured at 15 degrees C) of phosphatidylcholine from whole cells grown at 39 degrees C. On the other hand, both cardiolipins of 39 degrees C-grown and 15 degrees C-grown cells decreased the membrane fluidity of phosphatidylcholine from Tetrahymena cells grown at 15 degrees C. The same results were obtained for phosphatidylcholines of mitochondria and microsomes. Membrane fluidity of phosphatidylethanolamine, isolated from cells grown at 15 degrees C, was reduced to a small extent by Tetrahymena cardiolipin whereas that of 39 degrees C-grown cells was not changed. Representative molecular species of phosphatidylcholines of cells grown at 39 degrees C and 15 degrees C were synthesized chemically; 1-palmitoyl-2-oleoylphosphatidylcholine for 39 degrees C-grown cells and dipalmitoleoylphosphatidylcholine for 15 degrees C-grown ones. By the addition of Tetrahymena cardiolipin, the membrane fluidity of 1-palmitoyl-2-oleoylphosphatidylcholine was not changed but that of dipalmitoleoylphosphatidylcholine was decreased markedly. These phenomena were caused by Tetrahymena cardiolipin. However, bovine heart cardiolipin, which has a different composition of fatty acyl chains from the Tetrahymena one, exerted only a small effect.     

Solubilization, purification, and characterization of cardiolipin synthase from rat liver mitochondria. Demonstration of its phospholipid requirement.

Cardiolipin is a specific and functionally important phospholipid of mitochondria, and its biosynthesis is considered to be crucial for the assembly of this organelle. However, little information is available about the enzyme cardiolipin synthase, largely because it has not yet been isolated. We solubilized cardiolipin synthase from rat liver mitochondrial membranes with Zwittergent 3-14 and purified it by Mono Q anion exchange chromatography, Superose 12 gel filtration, and Mono P chromatofocusing. Cardiolipin synthase is one of the most acidic mitochondrial proteins (isoelectric point, pH 4-5) and appears as a 50-kilodalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme requires CO2+ for activity, has an alkaline pH optimum (pH 8-9), and exhibits Km values of 45 and 1.6 microM for phosphatidylglycerol and CDP-diacylglycerol, respectively. Cardiolipin synthase loses activity during purification, and the activity can be partially reconstituted by the addition of phospholipids. The most effective phospholipid is phosphatidylethanolamine which reactivates in a cooperative manner. Cardiolipin reactivates hyperbolically at low concentrations but inhibits the enzyme at higher concentrations. In addition, cardiolipin shifts the sigmoidal reactivation curve of phosphatidylethanolamine toward lower concentrations. It is suggested that cardiolipin synthase requires interaction with several molecules of phosphatidylethanolamine and at least one molecule of cardiolipin for full enzymatic activity.     

Archaebacterial lipid membranes as models to study the interaction of 10-N-nonyl acridine orange with phospholipids

The dye 10-N-nonyl acridine orange (NAO) is used to label cardiolipin domains in mitochondria and bacteria. The present work represents the first study on the binding of NAO with archaebacterial lipid membranes. By combining absorption and fluorescence spectroscopy with fluorescence microscopy studies, we investigated the interaction of the dye with (a) authentic standards of archaebacterial cardiolipins, phospholipids and sulfoglycolipids; (b) isolated membranes; (c) living cells of a square-shaped extremely halophilic archaeon. Absorption and fluorescence spectroscopy data indicate that the interaction of NAO with archaebacterial cardiolipin analogues is similar to that occurring with diacidic phospholipids and sulfoglycolipids, suggesting as molecular determinants for NAO binding to archaebacterial lipids the presence of two acidic residues or a combination of acidic and carbohydrate residues. In agreement with absorption spectroscopy data, fluorescence data indicate that NAO fluorescence in archaeal membranes cannot be exclusively attributed to bisphosphatidylglycerol and, therefore, different from mitochondria and bacteria, the dye cannot be used as a cardiolipin specific probe in archaeal microorganisms.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria II. Preferential interaction of cardiolipin with specific molecular species of phospholipid

A specific effect of cardiolipin on fluidity of mitochondrial membranes was demonstrated in Tetrahymena cells acclimated to a lower temperature in the previous report (Yamauchi, T., Ohki, K., Maruyama, H. and Nozawa, Y. (1981) Biochim. Biophys. Acta 649, 385–392). This study was further confirmed by the experiment using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Anisotropy of DPH for microsomal and pellicular total lipids from Tetrahymena cells showed that membrane fluidity of these lipids increased gradually as the cells were incubated at 15°C after the shift down of growth temperature from 39°C. However, membrane fluidity of mitochondrial total lipids was kept constant up to 10 h. This finding is compatible with the result obtained using spin probe in the previous report. Additionally, the break-point temperature of DPH anisotropy was not changed in mitochondrial lipids whereas those temperatures in pellicular and microsomal lipids lowered during the incubation at 15°C. Interaction between cardiolipins and various phospholipids, which were isolated from Tetrahymena cells grown at 39°C or 15°C and synthesized chemically, was investigated extensively using a spin labeling technique. The addition of cardiolipins from Tetrahymena cells grown at either 39°C or 15°C did not change the membrane fluidity (measured at 15°C) of phosphatidylcholine from whole cells grown at 39°C. On the other hand, both cardiolipins of 39°C-grown and 15°C-grown cells decreased the membrane fluidity of phosphatidylcholine from Tetrahymena cells grown at 15°C. The same results were obtained for phosphatidylcholines of mitochondria and microsomes. Membrane fluidity of phosphatidylethanolamine, isolated from cells grown at 15°C, was reduced to a small extent by Tetrahymena cardiolipin whereas that of 39°C-grown cells was not changed. Representative molecular species of phosphatidylcholines of cells grown at 39°C and 15°C were synthesized chemically; 1-palmitoyl-2-oleoylphosphatidylcholine for 39°C-grown cells and dipalmitoleoylphosphatidylcholine for 15°C-grown ones. By the addition of Tetrahymena cardiolipin, the membrane fluidity of 1-palmitoyl-2-oleoylphosphatidylcholine was not changed but that of dipalmitoleoylphosphatidylcholine was decreased markedly. These phenomena were caused by Tetrahymena cardiolipin. However, bovine heart cardiolipin, which has a different composition of fatty acyl chains from the Tetrahymena one, exerted only a small effect.     

Competitive interaction of serotonin and the dye acridine orange with DNA

The interaction of serotonin and acridine orange dye with DNA isolated from bacterium Escherichia coli and the yeast Candida utilis has been analysed by spectrofluorimetric method. Using data on competitive binding to DNA of serotonin and acridine orange, known as DNA intercalator, a conclusion concerning the formation of intercalated complex between serotonin and DNA has been made. It is shown that for yeast DNA the constant of intercalated binding of serotonin is 3,5-fold smaller than for the bacterial one.     

Molecular mechanical studies of proflavine and acridine orange intercalation.

Previous workers have reported that proflavine and acridine orange form various structurally different complexes with the dinucleoside phosphates rCpG and dCpG, with uniform C3'-endo and mixed C3'-endo (3'-5') C2'-endo sugar puckers being observed. We present theoretical calculations, based on the method of molecular mechanics, which support the experimental observations. The results suggest that the mixed C3'-edo (3'-5') C2'-endo pucker conformation isi intrinsically more stable than the uniform C3'-endo conformation, but that the additional stabilisation gained from specific, hydrogen bonding, interactions between nucleic acid and solvent, or intramolecularly within the nucleic acid, can lead to the adoption of the latter conformation, or of variants between the two. The role played by hydrogen bonding between amino-groups and nucleic acid phosphate appears more subtle than previously supposed.     

The differential fluorescence of bacteria stained with acridine orange and the effects of heat.

Some factors affecting the fluorescence of bacteria stained with acridine orange and the direct epifluorescent filter technique (DEFT) were studied. When bacterial cells from a chemostat operated at dilution rates between 0.1 and 0.7/h were used the differential fluorescence observed in the DEFT related to cell 'activity' and the orange fluorescence, which was predominant at high growth rates, may be related to an increase in the RNA content of the cells. Heat affected the colour of cell fluorescence and this was dependent on the cell type and, in particular, age. Uptake of acridine orange into the cells was also found to be an important factor determining the colour of fluorescence. However, with heat-treated cells there was no correlation between the amount of uptake and colour of fluorescence. The relative amounts and degree of denaturation of the different types of nucleic acids remaining in the cells after heat treatment appeared primarily to determine the colour of fluorescence.