GFP Fusion Proteins: A Solution or a Problem?

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GFP Fusion Proteins: A Solution or a Problem?

Journal Title: Biomedical Journal of Scientific & Technical Research (BJSTR) - Year 2018, Vol 5, Issue 5

Abstract

Using fluorescent proteins as imaging probes is a wide spread and versatile technique in microscopy. GFP tagged proteins can be used to track and examine real time localization, interactions and translocation of proteins of interest as well as to investigate several aspects of cell biology. The Y-box binding protein 1 (YB-1) is a pleiotropic protein involved in a widenumber of cellular processes and a substantial amount of knowledge on YB-1 localization and function was produced using YB-1-GFP constructs. Recently, it has been shown that YB-1 plays a critical role in cell stress response, playing an important role in the formation of stress granules (SG). To deeply investigate on this subject, we produced a stable HEK293T cell line constitutively expressing YB-1-GFP. In physiological conditions YB-1-GFP expressing cells behaved like the parental cells and YB-1-GFP protein properly localized to the cytoplasmic compartment. However, upon oxidative insult we observed a strong reduction in cell viability along with the occurrence of unusual GFP positive aggregates. Taken together, our findings suggest tocritically revise existing insights obtained with YB-1-GFP construct and, morein general, to beware and be critical in interpreting data obtained withfunctionally uncharacterized GFP fusion proteins.Y-box binding protein1 (YB-1) is a member of the evolutionarily conserved cold shock domain (CSD) proteins and was first identified as a DNA/RNA binding protein [1] involved in the control of gene expression at both transcriptional and translational level [2-4]. Given its multiple cellular functions, YB-1 is involved in the control of severalbiological processes including cell proliferation and migration. To properly perform its functions, YB-1 subcellular localization has to be finely regulated. Specific nuclear export (NES), nuclear localization (NLS) and cytoplasmic retention signals (CRS) contribute and direct the multifunctional tasking of YB-1 [5]. In normal resting cells, YB-1 localizes to cytoplasm where it is a majorcomponent of P-bodies and messenger ribonucleoproteins (mRNPs) [6]. Recent studies link YB-1 tothe cellular response tooxidative stress and DNA repair mechanisms. Indeed, following acute oxidative stress, YB-1 localizes to cytoplasmic Stress Granules (SGs), organelle-likestructures devoid of membranes engagedin mRNA sorting and pro-survival translational reprogramming [7,8]. In particular, YB-1 is recruited in TIA-containing Stress Granules (SGs) where it functions as a component of translationally inactive mRNPs todirectly block translational initiation of highly expressed [9]. Following DNA damage, YB-1 translocates to the nucleus andassociates with DNA repair protein complexes. Here we report the characterization of a stable pool of HEK293T cellsconstitutively ex pressing YB-1 as a GFP fusion protein enabling sensitiveanalysis of YB-subcellular localization by confocal microscopy.

Authors and Affiliations

Andrea Maria Guarino, Alessandra Pollice, Viola Calabro

Keywords

GFP; Stress Granules; Oxidative Stress; Stable Cell Line; Fluorescent Proteins Lipofectamine Trypsinized Stress Granules Transfection Cells Immunoblot Immunofluorescence Endogenous Humidified Antibiotics

EP ID EP588450
DOI 10.26717/BJSTR.2018.05.001258
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