Abstract
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.
Highlights
The localization of proteins to subcellular regions is closely related to protein function, and aberrant protein localization is a hallmark of many disease states such as cancer [1]
Fluorescence microscopy was used to determine enhanced green fluorescent protein (EGFP)-LC3 localization in amino acidstarved MCF-7 cells prior to measuring fluorescence intensity and lifetime by flow cytometry to ensure the presence of EGFP-LC3 punctae after starvation
The flow cytometry measurements obtained in this study in combination with fluorescence microscopy work suggest that the fluorescence lifetime of EGFP is sensitive to the formation of punctate EGFP distributions
Summary
The localization of proteins to subcellular regions is closely related to protein function, and aberrant protein localization is a hallmark of many disease states such as cancer [1]. The subcellular localization of a fluorescently labeled or fused target protein can be determined (i.e. association and disassociation with other proteins, presence in specific organelles, etc.). Using such methods has led to a more complete understanding with regard to the relationship between protein localization and function. Autophagy is a normal cellular process that is associated with various cell stresses This process can be induced by cell starvation, hypoxic conditions, or DNA damage. The LC3 protein is a critical component of the process of autophagy and the localization of LC3 to autophagosomes, specific organelles in a cell that form during autophagy, is a standard biomarker in autophagic cells. Autophagy acts as a source of energy during starvation conditions [19]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
