Membrane-targeted green fluorescent protein reliably and uniquely marks cells through apoptotic death
K J Harvey1, D Lukovic, D S Ucker
1Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
Background:
An understanding of the molecular processes that comprise the program of physiological cell death demands analytical techniques for the assessment of death events on the level of the individual cell, especially among transfectants and within heterogeneous populations. The utility of available transfection markers is limited by the variability of marker retention and discrimination as cells die. For example, soluble green fluorescent protein (GFP) leaks from dying cells and is not useful when fixation is required; conversely, transfected beta-galactosidase can be visualized only after fixation and staining.
Methods:
We have tested a GFP variant as a marker for the direct identification and visualization of transfected cells. We have explored the utility of this membrane-targeted GFP, the genetic fusion of the enhanced GFP and the farnesylation sequence of p21(Ras) (EGFP-F), in a variety of cell death assays.
Results:
EGFP-F is retained reliably in unfixed dying cells, permitting numerous events of the cell death process to be analyzed in real time in marked 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, independent of their ultimate fate. Cellular condensation of EGFP-F provides a specific and quantitative measure of physiological cell death.
