Abstract
Purpose: :
Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss Induced by Photobleaching (FLIP) are kinetic microscopy techniques that allow for the study of protein dynamics in living cells. The purpose of this study was to establish the dynamics of BCL2, an anti–apoptotic protein involved in epithelial renewal, in telomerase–immortalized human corneal epithelial cells (hTCEpi).
Methods: :
pEGFP expression plasmids were constructed for full length BCL2α, truncated BCL2 proteins at amino acid 546 abrogating the transmembrane domain (–TM), and site–directed BCL2 mutants at the serine 70 phosphorylation site (S70A). hTCEpi cells were transfected with pEGFP (control), pBCL2–EGFP, pBCL2–TM–EGFP, pS70A–EGFP, or pS70A–TM–EGFP. For live cell imaging, cells were maintained at 37°C and perfused with KGM culture media containing Hepes buffer. All experiments were performed using a Leica SP2 LSCM with 488 nm excitation. For FRAP XT analysis, scanning was performed bi–directionally at 1000 Hz, bleaching a 2 µm line ROI for 160 msec. Recovery was measured until a plateau was achieved. The amount of fluorescence recovery was calculated and the percent mobile fraction determined. For FLIP analysis, a bagel ROI was used to delineate the cytoplasmic compartment for bleaching and the nuclear compartment to monitor fluorescence loss. Images were acquired at 800 Hz, corrected for normal photobleaching effects, and the percent of protein translocation was determined.
Results: :
FRAP analysis showed that 2/3 of the protein for BCL2 and S70A is mobile in both compartments, with greater protein mobility in the cytoplasm than the nucleus. A reduction in protein mobility was seen in cells containing the (–)TM, accompanied by bright clustering of EGFP and followed by cell death. FLIP analysis demonstrated that mutation of the S70 regulatory phosphorylation site had no effect on translocation of BCL2 from the nucleus to the cytoplasm.
Conclusions: :
These preliminary findings suggest that: (1) a large fraction of these proteins are transiently dimerized and are not tightly bound to intracellular structures in situ; (2) TM mutations enhance rapid cell death; (3) translocation from nucleus to cytoplasm is not regulated by the S70 phosphorylation site. Further studies evaluating putative regulatory sites are in progress.
Keywords: cornea: epithelium • apoptosis/cell death • proteomics