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.

GFP Fusion Proteins: A solution or a problem?

Andrea Maria Guarino
Membro del Collaboration Group
;
Alessandra Pollice
Supervision
;
Viola Calabrò
Supervision
2018

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.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/731960
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