Open AccessSubcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry
Author(s) -
Ali Vaziri Gohar,
Ruofan Cao,
Patrick Jenkins,
Wenyan Li,
Jessica P. Houston,
Kevin D. Houston
Publication year - 2013
Publication title -
biomedical optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.362
H-Index - 86
ISSN - 2156-7085
DOI - 10.1364/boe.4.001390
Subject(s) - green fluorescent protein , subcellular localization , flow cytometry , protein subcellular localization prediction , confocal microscopy , fluorescence microscope , fusion protein , fluorescence , microbiology and biotechnology , confocal , intracellular , biology , chemistry , biophysics , biochemistry , cytoplasm , recombinant dna , gene , physics , geometry , mathematics , quantum mechanics
Intracellular protein transport and localization to subcellular regions are processes necessary for normal protein function. Fluorescent proteins can be fused to proteins of interest to track movement and determine localization within a cell. Currently, fluorescence microscopy combined with image processing is most often used to study protein movement and subcellular localization. In this contribution we evaluate a high-throughput time-resolved flow cytometry approach to correlate intracellular localization of human LC3 protein with the fluorescence lifetime of enhanced green fluorescent protein (EGFP). Subcellular LC3 localization to autophagosomes is a marker of the cellular process called autophagy. In breast cancer cells expressing native EGFP and EGFP-LC3 fusion proteins, we measured the fluorescence intensity and lifetime of (i) diffuse EGFP (ii) punctate EGFP-LC3 and (iii) diffuse EGFP-ΔLC3 after amino acid starvation to induce autophagy-dependent LC3 localization. We verify EGFP-LC3 localization with low-throughput confocal microscopy and compare to fluorescence intensity measured by standard flow cytometry. Our results demonstrate that time-resolved flow cytometry can be correlated to subcellular localization of EGFP fusion proteins by measuring changes in fluorescence lifetime.