Real-time analysis of endosomal lipid transport by live cell scintillation proximity assay

Abstract

A scintillation proximity assay has been developed to study the endosomal trafficking of radiolabeled cholesterol in living cells. Mouse macrophages were cultured in the presence of tritiated cholesterol and scintillant microspheres. Microspheres were taken up by phagocytosis and stored in phagolysosomes. Absorption of tritium beta particles by the scintillant produces light signals that can be measured in standard scintillation counters. Because of the short range of tritium beta particles and for geometric reasons, scintillant microspheres detect only that fraction of tritiated cholesterol localized inside phagolysosomes or within a distance of approximately 600 nm. By incubating cultures in a temperature-controlled microplate reader, the kinetics of phagocytosis and cholesterol transport could be analyzed in near-real time. Scintillation signals were significantly increased in response to inhibitors of lysosomal cholesterol export. This method should prove a useful new tool for the study of endosomal trafficking of lipids and other molecules.

Fig. 1

Theoretical probability distributions of the range of tritium β particles. Distributions were determined as described in Materials and Methods. The upper x axis represents distance in micrometers and the axis below represents energy in kiloelectron volts. The distance scale is related to the energy scale according to equation 1. All scales are decimal logarithmic. The solid line ordinates indicate the probability of tritium β particles having a kinetic energy greater than the corresponding abscissas on the keV scale as well as the probability of tritium β particles traveling farther in water than the corresponding abscissas on the μm scale. The dashed line was generated with equation 1 for a radius of 1.25 μm and indicates the probability of a linearly propagating particle reaching a sphere with a diameter of 2.5 μm as a function of the shortest distance between the particle’s origin and the sphere. The dotted line was generated by multiplication of discrete probability values from the preceding two data sets and gives an estimate for the probability of tritium β particles reaching a 2.5 μm sphere as a function of distance.

Fig. 2

Live cell scintillation proximity assay. A: J774 macrophages were set up at 2 × 10⁶ cells per well in medium B and supplemented with 6.5 μg/ml acetylated LDL {AcLDL; reconstituted with [1,2,6,7-³H(N)]cholesteryl oleate ([³H]CO); 160 μCi/mg protein} plus the indicated amount of yttrium silicate (YSi) beads per well. Proximity scintillation was recorded at 33°C over a period of 20 h. Each time point represents the average of two wells. Average coefficients of variation were 15% (7 μg/ml), 22% (20 μg/ml), 11% (70 μg/ml), and 3% (200 μg/ml). The inset shows the average proximity scintillation of the last five time points of each curve in A plotted as a function of the amount of beads per well (r² = 0.9986). SD values ranged from 3 to 34 cpm and are smaller than the symbols. B: J774 macrophages were set up at 2 × 10⁶ cells/well in medium B and supplemented with 100 μg of YSi beads plus the indicated concentrations of AcLDL (reconstituted with [³H]CO; 120 μCi/mg protein). Each time point represents the average of four wells. C: The average proximity scintillation of the last five time points of each curve in B is plotted as a function of the AcLDL concentration (r² = 0.9993). SD values varied from 14 to 50 cpm and are smaller than the symbols. D: For the 10 and 30 μg/ml AcLDL traces in B, the coefficients of variation are plotted as a function of the SD of each data point. The inset shows the average coefficient of variation (CV) for each curve in A.

Fig. 3

Phagocytosis dependence of in vivo proximity scintillation. A and B: On day 0, mouse peritoneal macrophages were set up at 10⁶ cells per well in medium A. On day 1, cells were switched to medium A plus 10 μg/ml AcLDL (reconstituted with [³H]CO; 120 nCi/mg protein) and incubated at 37°C for 24 h. On day 2, cells were switched to medium B plus 0.1% DMSO and 100 μM cytochalasin B (Cyto. B), 20 μM cytochalasin D (Cyto. D), or 0.5 μM latrunculin A (Lat. A) as indicated. After 30 min at 37°C, 150 μg of YSi beads was added to each well, and cells were incubated on ice for 30 min to allow the beads to settle. Subsequently, the dish was moved to a microplate scintillation counter at 33°C to initiate proximity scintillation readings. A: Time-resolved proximity scintillation of cells incubated in the absence or presence of cytochalasin D. Data points represent the average of four identically treated wells. Average coefficients of variation were 11% (control) and 16% (cytochalasin D). B: Proximity scintillation after 5 h in the presence of the indicated inhibitors. Error bars indicate SD (n = 5). C and D: On day 0, two dishes of J774 cells were set up at 25% confluence in medium A. On day 1, cells were switched to medium A plus [³H]CO-containing liposomes (1 nCi/ml medium) and incubated overnight. On day 2, 25 mg of scintillant polyvinyltoluene (PVT) beads were added to one dish and cells were allowed to phagocytose for 4 h. Then, cells were washed and harvested. Cells from the control dish were mixed with 25 mg of PVT beads after harvest. Cells were overlaid with a continuous 0–30% sucrose gradient and centrifuged for 2 h at 40,000 rpm. One milliliter fractions were collected, and total radioactivity and bead concentration were determined. C: Total radioactivity in individual fractions as determined by liquid scintillation counting. Dashed line, control; solid line, beads internalized by phagocytosis. The inset shows proximity scintillation in fraction 13 corrected for bead concentration (cpm/mg PVT). D: J774 cells were set up as in C and incubated with [³H]CO in the absence (Control) or presence (Prog.) of 10 μg/ml of progesterone as indicated. The cells were then incubated with PVT beads (6 mg/dish) for 4 h. Proximity scintillation and total radioactivity were determined before and after cell fractionation. Data represent the ratio of proximity scintillation in the peak bead-containing fraction versus proximity scintillation in intact cells before fractionation. Error bars indicate SD (n = 2).

Fig. 4

Effect of progesterone (Prog.) on proximity scintillation. A–C: J774 macrophages were set up at 10⁷ cells in 10 cm dishes in medium A. After 16 h, each dish received 1 mg of YSi beads, 10 μg/ml AcLDL (reconstituted with [³H]CO; 120 μCi/mg protein), and 0.1% ethanol with or without 10 μg/ml progesterone as indicated. After 16 h, cells were washed three times with PBS, scraped into 1 ml of PBS, and transferred to 20 ml scintillation vials. A: Proximity scintillation corrected for cellular protein concentration. B: After proximity scintillation readings, vials received liquid scintillation cocktail to determine total cellular radioactivity. C: Values represent the ratio of bead-derived scintillation in A divided by the total cellular radioactivity in B. Error bars indicate SD (n = 2). D: On day 0, two dishes of J774 cells were set up at 25% confluence in medium A. On day 1, cells were switched to medium A with [³H]CO-containing liposomes (1 nCi/ml medium) in the absence or presence of 10 μg/ml progesterone and incubated overnight. Cells then received 25 mg of PVT beads for 2 h. Subsequently, samples were subjected to 0–30% sucrose gradient centrifugation. Data indicate bead concentrations in individual gradient fractions. The inset shows total radioactivity normalized for bead concentration in fraction 9.