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Metabolism and intracellular localization of a fluorescently labeled intermediate in lipid biosynthesis within cultured fibroblasts.

In this paper we report on the uptake and distribution of an exogenously supplied fluorescent phosphatidic acid analogue by Chinese hamster fibroblasts. Under appropriate in vitro incubation conditions, 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidic acid was rapidly and preferentially transferred from phospholipid vesicles to cells at 2 degrees C. However, unlike similar fluorescent derivatives of phosphatidylcholine and phosphatidylethanolamine that remain restricted to the plasma membrane under such incubation conditions (Struck, D. K., and R. E. Pagano. 1080. J. Biol. Chem. 255:5405--5410), most of the phosphatidic acid-derived fluorescence was localized at the nuclear membrane, endoplasmic reticulum, and mitochondria. This was shown by labeling cells with rhodamine- containing probes specific for mitochondria or endoplasmic reticulum, and comparing the patterns of intracellular NBD and rhodamine fluorescence. Extraction and analysis of the fluorescent lipids associated with the cells after treatment with vesicles at 2 degrees or 37 degrees C revealed that a large fraction of the fluorescent phosphatidic acid was converted to fluorescent diglyceride, phosphatidylcholine, and triglyceride. Our findings suggest that fluorescent phosphatidic acid may be useful in correlating biochemical studies of lipid metabolism in cultured cells and studies of the Intracellular localization of the metabolites by fluorescence microscopy. In addition, this compound provides a unique method for visualizing the endoplasmic reticulum in living cells.

Small unilamellar vesicles were formed by ethanol injection (9). In a typical preparation, -1 pmol of lipids were mixed in thedesired proportions, dried under a stream of argon gas, and further dried in vacuo. The lipids were dissolved in 0.2 ml of ethanol, then injected into 2.6 ml of HCMF' with stirring . The preparation was then dialyzed at 4°C overnight against several changes of buffer. In some experiments, small vesicles were also prepared by ultrasonication (6), giving identical results.

Cell Culture and Vesicle-Cell Incubations
Monolayer cultures of Chinese hamster V79 fibroblasts (4) were grown to confluence in Eagle's minimal essential medium (EMEM) supplemented with 12% horse serum in a water-saturated atmosphere of 5% Co, in air. Single-cell suspensions were prepared by trypsinization as described (20) .
Cell pellets containing 10-20 x 106 cells were suspended in 1 ml of vesicle For lipid extraction, cell pellets were suspended in 0.9 ml of HCMF and acidified using 0.1 ml of IN HCI. The cell lipids were extracted with 3 ml of ethyl acetate:acetone (2 :1), followed by two additional extractions with 2.6 ml of ethyl acetate:acetone (2:0.6) (17) . The organic phase was then taken to dryness under a stream of argon gas, and the lipids were redissolved in CHCI3:CH,OH (2 :1) . The amounts of fluorescent NBD-(n.., = 470 nm ; Aem = 530 nm) and rhodamine-(X_ = 555 nm ; A .m = 585 nm) labeled lipids present in the extracts were determined by reference to standard curves generated from known amounts of NBD-PA or N-Rh-PE in CHCL,:CHa0H (2 :1) .
Lipid extracts were analyzed qualitatively by thin-layer chromatography on Silica Gel 60 thin-layer plates (Merck & Co ., Inc., Rahway, N. J.) in CHCI,,: CH;,OH :28% NH,OH (65:35:5). Plates were air-dried and the fluorescent lipids visualized for photography by excitation with UV light . For quantitative analysis of the fluorescent lipids, extracts were subjected to thin-layer chromatography as above, using silica gel H thin-layer plates (Analtech, Inc., Newark, Del.) . Fluorescent lipids were located with UV light, and the spots were scraped and the lipids extracted as described above, except that 1% Triton X-100 was present in the aqueous phase.

Microscopy
Microscopy was performed with a Zeiss Universal microscope equipped with Nomarski optics and epi-illumination for fluorescence . The light source for fluorescence was a tunable argon gas laser (Lexel Corp., Palo Alto, Calif) that was defocussed and attenuated for observation and photography. For NBD fluorescence, the sample was excited at 457.9 nm and fluorescence observed with an FL 500 dichroic mirror and BF 500 barrier filter suitable for the 530-nm emission peak of this fluorophore. For rhodamine fluorescence, the sample was excited at 514.5 nm and fluorescence observed with an FL 580 dichroic mirror and BF 580 barrier filter combination. The microscope was equipped with a micrometer stage to permit relocation of a given field of cells after initial photography and subsequent fixation and staining with a second fluorophore.

Intracellular Distribution of NBD-PA-derived Lipids
Monolayer cultures were incubated for 60 min at 2°C with NBD-PA-containing vesicles, washed with HCMF, and selected fields of cells were photographed . For subsequent staining of the endoplasmic reticulum in NBD-PA-treated cells the method of Virtanen et al . (21) was used, with modifications. After selected fields of NBD-PA-treated cells were photographed, the monolayer cultures were incubated for 30-45 min at 2°C with 100 I+g of nonfluorescent Lens culinaris agglutinin (Vector Laboratories, Burlingame, Calif.) per ml phosphate-buffered saline (PBS) (to block any surface receptors for the lectin) and washed with PBS. The cultures were fixed for 60 min at room temperature in 3% formaldehyde (3), washed with PBS containing 0.1 M glycine, and rinsed again with PBS. The cells were permeabilized by treatment with 0.1% Triton X-100 in PBS for 3 min (5) and thoroughly washed . Cells were then stained with 100 Ag of rhodamineconjugated Lens culinaris agglutinin (Rh-LcA; Vector Laboratories) per ml PBS for 20 min at room temperature, washed, and fixed for an additional 30 min. The stained cells were washed and photographed for rhodamine fluorescence . After this treatment, essentially no NBD fluorescence could be detected.
Mitochondria were visualized within cells using the fluorescent dye Rhodamine 3B (Eastman) as described by Johnson et al . (7). Monolayer cultures were incubated with 1 pg dye per ml at 2'C for 2 min, washed, and photographed using optics described above for rhodamine fluorescence.

RESULTS
When DOPC vesicles containing NBD-PA or NBD-PC and the nonexchangeable lipid, N-Rh-PE (12,19,20) were incubated with Chinese hamster fibroblasts at 2°C, significant amounts of the NBD-lipids were transferred to the cells with relatively little uptake of N-Rh-PE (Table I) . In every experiment, the ratio of NBD to rhodamine fluorescence in the washed, vesicle-treated cells was significantly greater than in the applied vesicle suspension . This demonstrates that the uptake of vesicle lipids was due to preferential exchange or transfer of lipids (13,14,16,20) and was not due to association of intact vesicles with cells . Uptake of intact vesicles would cause the ratio of NBD to rhodamine fluorescence in the cell extracts to be identical to that found in the starting vesicles. Whereas the amount of NBD-PC that became cell-associated was approximately linearly dependent on the mole fraction of NBD-PC present in the vesicles, a fourfold increase in NBD-PA concentration in the vesicles resulted in approximately a 14-fold enhancement in uptake of NBD-fluorescence by the cells. Vesicle-cell incubations at 37°C also resulted in preferential uptake of NBD-PA relative to N-Rh-PE (data not shown) . Finally, in control experiments it was found that the presence ofN-Rh-PE had no effect on the transfer of NBD-PA or NBD-PC from vesicles to cells . Fig . 1 and Table II     the NBD-PA was metabolized, with the majority being converted to NBD-DG, but also with small amounts of NBD-PC and 1,3-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl triglyceride (NBD-TG) being formed . At 37°C, the qualitative pattern of fluorescent products was unchanged but, quantitatively, significantly more fluorescent phosphatidylcholine and triglyceride were made. Vesicle-cell incubations were also carried out at 2°and 37°C using isotopically labeled phosphatidic acid in place of NBD-PA. As with NBD-PA, this compound was also hydrolyzed to diglyceride and further metabolized to phosphatidylcholine and triglyceride (unpublished observations) .

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Distribution of Cell-associated NBD-PA-derived Lipids Cells incubated with NBD-PA-containing vesicles at 2°C (Figs . 2 and 3) had little or no fluorescence at the cell surface. Instead, they had a bright fluorescence around the nucleus and substantial cytoplasmic fluorescence . Similar fluorescence was seen in both cell suspensions (Fig. 2) and monolayer cultures. In the monolayer cultures this fluorescence could be resolved into bright dots and a fine reticular network particularly visible at the cell periphery (Fig. 3) . This pattern of fluorescence persisted for hours as long as the cells were maintained at 2°C. When incubations were carried out at 37°C with NBD-PA, essentially identical patterns of labeling were seen except that the specimen was more intensely fluorescent because of the enhanced uptake at 37°C.
To identify the locations of NBD fluorescence, we first treated monolayer cultures of V79 cells with NBD-PA vesicles at 2°C and photographed selected fields of cells . Then, the endoplasmic reticulum in the same cells was visualized by labeling with Rh-LcA (21) (Fig. 4). Comparison of the NBD fluorescence (Fig. 4a, c) with the Rh-LcA fluorescence (Fig.  4b, d) demonstrated that the reticular network visualized by NBD-fluorescent lipids appears coincident with the endoplasmic reticulum .
We attempted to carry out similar colocalization experiments on NBD-PA-treated cells using the mitochondria-specific dye . Rhodamine 3B (7) but were unsuccessful . Apparently, photodamage that occurred during photography of the NBD-fluorescence inhibited subsequent uptake of the rhodamine dye by the photographed (but not by the surrounding) cells . Therefore, to determine whether mitochondria were labeled by treatment of cells with NBD-PA, we conducted separate experiments in which the fluorescence and Nomarski images of a given cell were compared. In Fig . 5 a it is seen that the Rhodamine 3Blabeled mitochondria correspond to cytoplasmic images that appear as bumps with Nomarski optics . In Fig . 5 b the fluorescent dots seen in NBD-PA-treated cells also correspond to such Nomarski images. We thus concluded that NBD-PA treatment of cells also leads to labeling of mitochondria .

DISCUSSION
In this report we have shown that an exogenously supplied fluorescent phosphatidic acid derivative, NBD-PA, was incorporated into mammalian fibroblasts in a distinctive manner. Whereas fluorescent analogues such as NBD-PC and NBDphosphatidylethanolamine label predominantly the cell surface during vesicle-cell lipid transfer at 2°C (20), under identical incubation conditions NBD-PA labeled the nuclear membrane, mitochondria, and endoplasmic reticulum. Furthermore, unlike NBD-PC or NBD-phosphatidylethanolamine, NBD-PA was converted to the fluorescent products expected from the established lipid biosynthetic pathways in mammalian cells. This metabolism of NBD-PA suggests that the presence of the fluorescent group on the acyl chain of the PA molecule does not inhibit the activity of enzymes involved in PA metabolism.
The intracellular localization of the NBD-lipids derived from exogenously supplied NBD-PA is of considerable interest. For determining the distribution of the fluorescent lipids within cells, we did not use cell fractionation methods because the NBD-lipid molecules might redistribute between membranes (I1, 12) during the fractionation procedure, and because pure subcellular membrane fractions are difficult to obtain . Instead, we used separate rhodamine-containing probes for mitochondria (7) and endoplasmic reticulum (21) and showed that the intracellular distributions of rhodamine and NBD fluorescence were highly correlated . Although correlation of NBD-fluorescence and the Rh-LcA-stained endoplasmic reticulum was not perfect, it should be noted that the rhodamine-fluorescence photomicrographs were made using fixed and permeabilized cells. Apparently, these procedures, which are necessary for staining of the endoplasmic reticulum with Rh-LcA, damaged the reticular network to some extent .
Although the presence of the NBD-lipids on the endoplasmic reticulum and nuclear membrane (an extension of the endoplasmic reticulum) might be expected because most of the enzymes involved in lipid biosynthesis are found there, the RAPID COMMUNICATIONS