Membrane-targeted green fluorescent protein reliably and uniquely marks cells through apoptotic death

Background: An understanding of the molecular processes that comprise the p rogram of physiological cell death demands analytical techniques for the as sessment of death events on the level of the individual cell, especially am ong transfectants and within heterogeneous populations. The utility of avai lable transfection markers is limited by the variability of marker retentio n and discrimination as cells die. For example, soluble green fluorescent p rotein (GFP) leaks from dying cells and is not useful when fixation is requ ired; conversely, transfected beta -galactosidase can be visualized only af ter fixation and staining. Methods: We have tested a GFP variant as a marker for the direct identifica tion and visualization of transfected cells. We have explored the utility o f this membrane-targeted GFP, the genetic fusion of the enhanced GFP and th e farnesylation sequence of p21(Ras) (EGFP-F), in a variety of cell death a ssays. Results: EGFP-F is retained reliably in unfixed dying cells, permitting num erous events of the cell death process to be analyzed in real time in marke d cells. Moreover, the cell rounding and shrinkage associated with the loss of adhesion during cell death result in a characteristic condensed EGFP-F signal. Conclusions: EGFP-F serves to identify transfectants consistently, independ ent of their ultimate fate. Cellular condensation of EGFP-F provides a spec ific and quantitative measure of physiological cell death. Cytometry 43:273 -278, 2001. (C) 2001 Wiley-Liss, Inc.