Vital Staining of Cells with Fluorescent Lipids
Although a number of fluorescent lipid analogs are now available, only a limited set of the molecules are frequently employed for cell biology. Because most of these molecules are less hydrophobic than naturally occurring counterparts, they are easily partitioned to cell membranes when added to the medium. This property and bulk fluorophore alter both physical and biological properties of the lipids. Therefore, the intracellular behavior of fluorescent lipids is not always the same as that of endogenous lipids. In this context, the results need to be evaluated carefully. However, together with appropriate control, fluorescent lipids are very useful tools to follow dynamics of membrane lipids.
II. MATERIALS AND INSTRUMENTATIONS
6-((N- (7-nitrobenz-2-oxa- 1,3-diazol-4-yl) amino) hexanoyl)sphingosylphosphorylcholine (C6-NBD-SM, MW 740.88, Cat. No. N3524), 6-((N-(7-nitrobenz-2- oxa-l,3-diazol-4-yl)amino)hexanoyl)sphingosine (C6- NBD-Cer, MW 575.75, Cat. No. Nl154) and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indaene-3-pentanoyl)sphingosine (C5-BODIPY-Cer, MW 601.63, Cat. No. D3521) are from Molecular Probes. 12- ((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)dodecanoyl) sphingosine (C12-NBD-Cer, MW 659.90, Cat. No. N9158) is from Sigma. 1-Palmitoyl-2-(6-((7-nitro-2- 1,3-benzoxadiazol-4-yl)amino)hexanoyl)-sn-glycero-3-phospho-L-serine (ammonium salt) (C6-NBD-PS, MW 790.85, Cat. No. 810192) is obtained from Avanti. Structures of fluorescent lipids are summarized in Fig. 1.
Dulbecco's modified Eagle's medium (DMEM, Cat. No. 11885-084) is obtained from Invitrogen. Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham, without phenol red (DMEM F-12, Cat. No. 6434), is from Sigma. Thirty-five-millimeter glass-bottom culture dishes are from Matsunami Glass (noncoat, Cat. No. 111100) or from IWAKI (Asahi Technoglass Scitech Division IWAKI brand, Cat. No. 3911-035) and 60-mm culture dishes are from Nunc. Dulbecco's phosphate-buffered saline (-) (PBS, Cat. No. 14249-95) and sodium dithionite (sodium hydrosulfite, Cat. No. 31509-55) are from Nacalai Tesque. Brefeldin A (Cat. No. G-405) is from BIOMOL. The stained cells are examined under a Zeiss LSM 510 confocal microscope equipped with C-Apochromat 63XW Korr (1.2n.a.) objective.
III. PROCEDURES A. Fate of Different NBD-Labeled Lipids Introduced into Plasma Membranes of Cultured Fibroblasts
This procedure describes the methods used to follow the fate of fluorescent lipids inserted into the outer leaflet of the plasma membranes. The fate of fluorescent lipids is different depending on the hydrophilic head group of the lipids.
Solutions of Fluorescent Lipids
Stock solutions (1 mg/ml) of fluorescent lipids are prepared by dissolving C6-NBD-PS in chloroform and C6-NBD-SM in ethanol. The chloroform stock solution should be kept in screw-capped (Teflon-lined) glass tubes. The ethanol solution can be stored in either glass or plastic tubes. Stock solutions are stored at -20°C. An aliquot of a stock lipid solution is dried, first under a stream of nitrogen and then in vacuo. The dried lipid is then dissolved in DMEM to give final concentration 4µM. The solution is vortexed and kept at 4°C before use.
C6-NBD-SM (Koval and Pagano, 1989) and C6- NBD-PS (Martin and Pagano, 1987) were first introduced by Pagano and co-workers. At low temperature (2 or 4°C), both fluorescent lipids are transported from the medium to the outer leaflet of the plasma membranes of cultured cells via spontaneous transfer of the lipids. Warming up of cells induces ATP-dependent transbilayer movement (flip-flop) of C6-NBD-PS (Martin and Pagano, 1987). In mammalian cells, the energy-dependent transbilayer movement of lipids is observed only with amino phospholipids such as phosphatidylserine and phosphatidylethanolamine (Devaux et al., 2002). Flip-flop is observed as low as 7°C. Once internalized, C6-NBD-PS is transported spontaneously to intracellular membranes. Unlike C6- NBD-PS, C6-NBD-SM stays on the outer leaflet of the plasma membrane and is internalized by endocytosis. It is postulated that C6-NBD-SM is a marker for the bulk flow of membranes after endocytosis (Mayor et al., 1993). C6-NBD-SM is also used together with C6- NBD-labeled glycolipids to study polarized lipid transport in hepatoma cells (Maier et al., 2002). Because DMEM F-12 contains HEPES, the pH keeps constant during incubation outside the CO2 incubator. We use phenol red-free DMEM F-12 when we take images under a microscope. BODIPY-labeled lipids (Pagano and Chen, 1998) and pyrene-labeled lipids (Somerharju, 2002) are also used for the similar purpose. The fluorescent cholesterol analog dihydroergosterol is introduced to follow the fate of cell surface cholesterol (Mukherjee et al., 1998).
B. Measurement of Recycling of NBDLabeled Sphingomyelin Using Dithionite
The dithionite method is a simple procedure used to bleach NBD located outside cells. With this procedure, it is possible to quantitate the appearance of NBD-lipid to the plasma membrane.
1. Cellular Distribution of Fluorescent Lipids
The fluorescent lipid solution is prepared as described in Section III,A. Sodium dithionite solution is prepared by diluting a freshly made 1M aqueous stock solution with DMEM (final concentration: 50 mM).
2. Quantitation of Recycling of C6-NBD-SM
Selective removal of fluorescent lipid present on the outer leaflet of the plasma membrane allows the quantitative analysis of the kinetics of lipid transport to the plasma membrane. This is achieved by extracting C6-NBD- and C5-BODIPY-labeled phospho- and sphingolipids with BSA (Sleight and Pagano, 1984). Alternatively, one can selectively destroy NBD-labeled lipids on the outer monolayer with the water-soluble quencher dithionite (McIntyre and Sleight, 1991). The dithionite method is widely used to monitor transbilayer distribution (Nolan et al., 1995; Williamson et al., 1995) and vesicular traffic (Babia et al., 2001), as well as lateral mobility (Kobayashi et al., 1992), of NBDlabeled lipids. However, because dithionite can leak through the membrane in some cells, proper conditions have to be adapted to each specific case (Pomorski et al., 1994). Recycling of C6-NBD-SM is very rapid (Hao and Maxfield, 2000). The distribution of C6-NBD-SM shown here is the result of several recycling rounds of the fluorescent lipid. Internalized C6-NBD-SM is also transported to lysosomes and metabolized to C6-NBD-Cer. Transport to the degradative compartments is estimated to be 18- to 19-fold slower than the rate of C6-NBD-SM recycling (Koval and Pagano, 1990). In some cells, degradation of C6-NBD-SM occurs on the plasma membrane in a differentiation-dependent manner (Kok et al., 1995). Such degradation products are separated from C6- NBD-SM by thin-layer chromatography (Kobayashi and Pagano, 1989; Koval and Pagano, 1990).
C. Vital Stain of Golgi Apparatus by Fluorescent Ceramide Analogs
Fluorescent ceramide is widely used for vital staining of the Golgi apparatus (Pagano, 1989; Pagano et al., 1991). Dynamics of the Golgi membranes could be followed by using fluorescent ceramide.
1. Vital Stain of Golgi Apparatus by Fluorescent Ceramides
Stock solutions (1 mg/ml) of fluorescent lipids are prepared by dissolving C6-NBD-Cer, C12-NBD-Cer, and C5-BODIPY-Cer in ethanol. An aliquot of a stock lipid solution is dried, first under a stream of nitrogen and then in vacuo. The dried lipid is dissolved in DMEM to give a final concentration 0.5 µM (C6-NBDCer and C5-BODIPY-Cer) or 5 µM (C12-NBD-Cer). The solutions are vortexed and kept at 4°C before use.
2. Alteration of Morphology of Golgi Apparatus Monitored by C5-BODIPY-Cer
Lipid solutions are prepared as described in Section III,C,1. 5-mg/ml stock solution of brefeldin A is diluted with DMEM F-12 to give a final concentration 5 µg/ml.
Three different fluorescent ceramides are now available commercially. All three give similar Golgi labeling, although their working concentrations are different. Mechanisms of the accumulation of NBDlabeled ceramide and BODIPY-labeled ceramide are apparently different (Martin et al., 1993). Fluorescent ceramide makes it possible to follow the fate of Golgi lipids in living cells under various conditions such as brefeldin A treatment (Lippincott-Schwartz et al., 1991). Brefeldin A treatment induces redistribution of the Golgi apparatus into the endoplasmic reticulum (Chardin and McCormick, 1999). In Fig. 6, we adjusted the laser power of the confocal microscope to prevent bleaching of the fluorescence during observation. Spectral properties of BODIPY-labeled ceramides also allow us to differentiate membranes containing high concentrations of the fluorescent lipid and its metabolites from other regions of the cell where smaller amounts of the probe are present with a specific filter setup (Pagano et al., 1991).
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