Morphological Detection of Plasma Membrane Changes During Apoptosis Using Enhanced Green Fluorescent Protein

Apoptosis, or programmed cell death, is one of the most widely studied areas of research today. The cellular alterations that occur during apoptosis have been well characterized. In fact, morphological changes to cells formed the basis of the original descriptions of apoptosis (7,8). The hallmarks of apoptotic cell death include cell shrinkage, plasma membrane changes and nuclear fragmentation (16,17). Changes in the plasma membrane have been widely observed during apoptosis, and these events are thought to be instructive for understanding how macrophages identify apoptotic cells. One of the earliest plasma membrane changes that occurs in response to apoptotic stimuli is the externalization of phosphatidylserine from the inner leaflet onto the outer membrane (3). The next series of events includes the formation of protrusions in the plasma membrane, which is usually referred to as membrane blebbing. Eventually the cell dissociates into membrane-bound particles that are referred to as apoptotic bodies. Finally, the apoptotic bodies are recognized and engulfed by macrophages (7). These changes are most often assessed using light or electron microscopy. In this study, we describe a novel noninvasive method to visualize plasma membrane changes that occur during apoptosis using fluorescence microscopy. We use a vector (pEGFP-F; CLONTECH Laboratories, Palo Alto, CA, USA) that targets enhanced green fluorescent protein (2,5,11) to the plasma membrane (1,6). Expression of EGFP-F results in a fluorescent green plasma membrane, allowing clear detection of morphological changes and providing the added benefit of marking transfected cells. We used EGFP-F to trace the progression of morphological changes that occur in the plasma membrane in response to apoptotic stimuli. To visualize plasma-membrane morphological changes in response to apoptotic stimuli, we transfected HeLa cells with pEGFP-F (4 μg/mL) using liposome-mediated transfection (19). EGFP-F contains the 20-amino acid farnesylation signal from c-Ha-Ras added to the C terminus of EGFP (6). The Ras farnesylation signal has been used in many studies to target heterologous proteins to the plasma membrane (1,4). HeLa cells that were successfully transfected with EGFP-F were identified by their green fluorescent plasma membrane. After transfection, but before the addition of apoptotic stimuli, the HeLa cells appeared flat (Figure 1A). Transfection with EGFP-F did not have an effect on the morphological appearance of the cells (Figure 1A). The cells were induced to undergo apoptosis by addition of 1 μM staurosporine (CLONTECH). Within 30 min after staurosporine addition, the cells began to appear round (Figure 1B). This change was readily detected due to the morphological changes emphasized by the fluorescent green membrane. Next, membrane blebbing was observed, and the borders of the bubble-like projections were accentuated due to EGFP fluorescence (Figure 1C). When the cell dissociated into apoptotic bodies (Figure 1, D and E), small membrane fragments were still visible. These morphological changes were clear and distinct due to the plasma membrane fluorescence from EGFP-F. Standard methods use phase contrast or differential interference contrast (DIC/Nomarski) light microscopy to follow these changes. One advantage EGFP fluorescence offers over these methods is detection of the most subtle plasma membrane changes that would not be detected otherwise. However, electron microscopy is the best method to use for detailed study of apoptosis-induced ultrastructural changes. Another advantage of using EGFP fluorescence is resistance to photobleaching—it has been shown to be more resistant to photobleaching than fluorescein (10,15). One limitation of the method we describe is that it involves transfection of cells with EGFP-F. In studies that require minimal cellular manipulation, an alternative method of detecting apoptotic cells should be used. In this context, plasma membrane changes can be examined using less invasive methods such as staining the cells with annexin V-fluorescein isothiocyanate (FITC) (12–14,18). The annexin V moiety binds to the phosphatidylserine exposed on apoptotic cells, whereas the FITC conjugate allows fluorescent detection of these cells. The ability to track plasma membrane changes offers many advantages. EGFP-F can be used in co-transfection experiments to determine the ability of transfected genes to inhibit or induce apoptosis. In previous studies, cytoplasmic EGFP had been co-transfected with known apoptosis-inducing genes, tumor necrosis factor-R (TNF-R) and Fas (9), to identify positively transfected cells. It was reported that the cotransfection of EGFP had no negative cellular effects, while it provided a convenient method to identify transfected cells (9). In our experience, we found Benchmarks