Metabolic incorporation of 9-(2-anthryl)-nonanoic acid, a new fluorescent and photoactivable probe, into the membrane lipids of Chinese hamster ovary cells

Abstract

-EJB 850836 9-(2-Anthryl)-nonanoic acid is a new fluorescent and photoactivable probe, which has been designed for studying the lateral diffusion rate and the lateral distribution of lipids in biological membranes by means of the anthracene photodimerization reaction. It is shown that this anthracene fatty acid is metabolically incorporated into the glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol) of the eukaryotic Chinese hamster ovary cells in culture.Under our culture conditions (Eagle's minimal essential medium plus delipidized fetal calf serum) this incorporation proceeded with a very good rate (up to 45 mo1/100 mol, after two days culture) and could be easily modulated depending on the way the cells were fed with the anthracene fatty acid. It occurred to a similar extent at the sn-1 (55 & 5%) or at the sn-2 (45 f 5%) position on the phospholipid glycerol backbone, without any degradation or elongation. No double labelling at the sn-1 and sn-2 positions was detected. Although incorporation of the anthracene fatty acid affected the cell growth rate (generation time of 48 h compared to a generation time of 21 h for control cells) it did not bring about cell mortality. This incorporation took place not only into the phospholipids but also into the triglycerides with, as a consequence, the appearance of strongly fluorescent lipid vesicles inside the cells. It affected the whole cell fatty acid composition by slightly increasing the amount of palmitic acid and markedly decreasing the amount of stearic and oleic acids.Controlled alterations of membrane lipid composition in eukaryotic cells [l] Recently, in order to go further in the description of membrane structure and to be able to investigate both the lateral distribution of lipids at a 'microscopic' level, in terms of neighbourhood relationships, and their dynamics (lateral diffusion rate), we have proposed a new photochemical approach, which uses anthracene as a photoactivable group, after it has been attached to a fatty acid [18]. This hydrophobic group, which is well suited for labelling the hydrophobic core of the membrane, is fluorescent and under illumination (360 nm) it forms 9-9', 10-10 covalently bound dimers, which are no longer fluorescent [19]. Thus, after its incorporation into the membrane lipids, this group has potential uses (a) as an indicator of membrane fluidity [20]; (b) for measuring the lateral diffusion rate of the labelled molecules by using the technique of fluorescence recovery after the photodimerization reaction [21] ; (c) for studying the topological distribution of lipids in membranes by means of a photo-cross-linking reaction between adjacent anthracene-labelled molecules (dimerization) and identification of the photodimers. Esters of 9-anthracene carboxylic acid, with fatty acids hydroxylated at various positions, have already been described as extrinsic fluorescent probes in model and natural membranes [20]. Nevertheless, these anthroyloxy fatty acids are not the ...

The spatial distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus varies during the cell cycle

“…The culture medium was supplemented with fatty acid-free bovine serum albumin (1 mg/ml) to favor dispersion of the anthracene fatty acid [15]. 3H-ANno (spec.…”

“…synchronously dividing cells For photodimerization experiments, cells were grown in the presence of radioactive anthracene fatty acid from the beginning to the end of the culture. The culture medium was supplemented with fatty acid-free bovine serum albumin (1 mg/ml) to favor dispersion of the anthracene fatty acid [15]. 3H-ANno (spec.…”

“…Lipids labeled with ANno display phase and physical properties similar to those of normal lipids [13]. As another advantage, ANno can be incorporated into the lipids of prokaryotic [14] and eukaryotic [15] cells at a high rate via regular lipid metabolic pathways, thus enabling constitutive lipids to be studied in situ. In the membrane of the gram-positive bacterium Micrococcus luteus, this approach coupled with a study of the transverse distribution of lipids revealed the existence of heterogeneities in the lateral distribution of the two major lipids, namely phosphatidylglycerol and dimannosyldiacylglycerol, [16].…”

Transverse and lateral distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus

“…The polymethylenic chains are only slightly influenced by the presence of anthracene with its long axis parallel and attached at the w-position of nonanoic acid (de Bony & Tocanne, 1982). 9-AN is incorporated at a high rate into the lipids of prokaryotic (Welby & Tocanne, 1982) and eukaryotic cells (Dupou et al, 1986) via normal metabolic pathways. Various anthracene phospholipid derivatives have been synthesized, and a new photo-cross-linking method has been devised for the identification and analysis of the photoproducts in various model membranes .…”