As proof of principle, pHMA was expressed in Drosophila cultured cells and its localization and fluorescence characteristics were monitored over the life-cycle of the cells. S2 and S2R+ cells, which are hemocyte-like, phagocytic cells derived from late-stage Drosophila embryos, were used for this analysis (Stroschein-Stevenson et al., 2006). To establish the relationship between intracellular pH and pHMA's fluorescence response, pHMA-expressing cells were fixed and permeabilized and the pH was adjusted with phosphate buffers ranging from pH 4.5 to 8.5, in 0.5-pH intervals. The ratio of pHMA emission when excited by 470 and 410 nm light (R470/410) was determined at each pH interval (Fig. 2A). Nonlinear regression analysis was used to generate a standard curve of pHMA R470/410 relative to pH (r2 = 0.998). The resulting model was used to approximate the pH of individual living, dying, and engulfed cells observed during time-lapse recordings (Fig. 2B). Time-lapse recordings of S2 cells that were transfected with Ubi-pHMA for two days were made over 24-hr periods with images taken at 10-min intervals. Over 400 S2 cells transiently expressing pHMA were analyzed. During the 24-hr imaging period, 48 pHMA-expressing cells (11%) were engulfed, while 30 (7%) suddenly disappeared, suggesting a necrotic demise. The number of engulfed pHMA-positive cells did not appreciably accumulate from the beginning 31/418 (7%) to the end of the recording period 40/443 (9%), indicating that the corpses were completely processed over time, and that the exposure conditions of the 10-min time-lapse cycle did not induce photo-toxic apoptosis. The pH of cells that were morphologically healthy stayed between 7.0–7.4 (cell a: Fig. 2B–D, C′, D′; see Supp. Movie 1, which is available online). Most, but not all, of these cells had cortically localized pHMA. Healthy cells displayed variable morphology that ranged from highly crenated to relatively round with long processes. Cells that were eventually engulfed or ruptured displayed decreased motility, rounded up, and the expressed pHMA frequently transitioned from cortical to cytoplasmic. These changes were accompanied by slight acidification to 6.8–6.6. Acidification of cytoplasm has previously been described as a component of apoptosis (Barry and Eastman, 1992). For example, in U937 cells the pH has been observed to vary from 7.2 in normal to 6.8 in pre-apoptotic to 5.7 in apoptotic cells (Nilsson et al., 2003, 2006). About 40% of the slightly acidified cells rapidly and completely lost their pHMA signal (cell b: Fig. 2B, E–G, E′–G′; Supp. Movie 2); this loss is presumed to result from the loss of membrane integrity since the non-fluorescent, poorly refractile cell corpses were still visible after the loss of pHMA signal, indicating necrosis. The frequency of cell rupture was greatly increased by adding cell death inducing agents such as 10 μM actinomycin D (data not shown). Cells that were engulfed, acidified over 30–60 min to a much greater degree, pH <6, than pre-engulfment. Most engulfed corpses remained acidified at pH 5 or below for several hours, while the phago-lysosome decreased in size over time (cell c: Fig. 2B, H–K, H′–K′; Supp. Movie 3). The pH of the phago-lysosomes appeared to rebound; most cell corpses partially re-neutralized (cells d and e: Fig. 2B, L–O, L′–O′; Supp. Movie 4). The biological significance of this phagosome pH recovery is not clear at this point, but will require further examination. All of the dying cells were engulfed whole. Over the 24-hr recording period, we observed approximately 140 partial engulfment events that possibly involved the pruning of cell processes from healthy cells or the engulfment of detached cell fragments. As will be described in the in vivo analysis of pHMA, this wholesale engulfment of dying cells by S2 cells differs from the piecemeal engulfment of epithelial cells seen in live embryos. This may be due to the experimental conditions or to the hemocytic nature of S2 cells. These data clearly show that pHMA-expressing S2 cells provide a useful platform for studying cell engulfment in vitro.
Figure 2. pHMA acidifies after cell engulfment in S2 cells. A: Standard R470/410 curve generated by fixing and buffering S2R+ pHMA-expressing cells. The average R470/410 and standard deviations (see Experimental Procedures section) are plotted against pH. B: pH estimates for cells “a” to “e” derived from the curve fit in A. C–O: Wide-field fluorescence images of cells from time-lapse movies; emission at excitation of 410 nm is green and at excitation of 470 nm is red. C′–O′: Fluorescence images are overlaid with transmitted light images to reveal unlabeled cells in the field. C,D, C′,D′: Morphologically healthy cell has cortical expression of pHMA, cell “a” (arrow). E–G, E′–G′: Dying cell “b” (arrow) acidifies slightly (greenyellow); fluorescence disappears. H–K, H′–K′: Cell “c” acidifies slightly and then acidifies substantially after engulfment and persists for many hours as a strongly acidified, but shrinking vesicle. L–O, L′–O′: Cells “d” and “e” acidify slightly and then acidify substantially after engulfment by “f′” and “f′”, the daughter cells of “f.” Notice that the pH of “d” and “e”'s engulfed corpses rebounds over time. Scale bar = 10 μm.
Download figure to PowerPoint