LET THERE BE LIGHT: A SENSITIVE LUCIFERASE ASSAY FOR QUANTIFYING CELL-CELL FUSION

0580784 - ÚMG 2024 RIV CZ eng A - Abstrakt
Trávníček, Martin - Štafl, Kryštof - Jech, Lukáš - Hejnar, Jiří - Trejbalová, Kateřina
LET THERE BE LIGHT: A SENSITIVE LUCIFERASE ASSAY FOR QUANTIFYING CELL-CELL FUSION.
Czech Chemical Society Symposium Series. Roč. 21, č. 5 (2023), č. článku P-65. ISSN 2336-7202.
[Annual meeting of the National Institute of Virology and Bacteriology (NIVB) /2./. 02.10.2023-05.10.2023, Kutná Hora]
Grant CEP: GA MŠMT(CZ) LX22NPO5103
Institucionální podpora: RVO:68378050
Klíčová slova: syncytin-1 and syncytin-2 * placental cells * cell-cell fusion assessmen
Obor OECD: Biochemistry and molecular biology
http://ccsss.cz/index.php/ccsss/issue/view/41

The successful formation of the mammalian placenta depends on the expression of genes of retroviral origin. Among these genes are human syncytin-1and syncytin-2, which encode retroviral envelope glycoproteins capable of triggering the fusion of phospholipid membranes. During evolution, both Syncytins were repurposed to facilitate the fusion of placental cells, introducing a novel physiological role. Within the placental cytotrophoblast, Syncytin-1 employs the neutral amino acid transporter ASCT2 as its specific membrane receptor1, while Syncytin-2 engages with the lysophosphatidylcholine transporter MFSD2a2. These interactions induce a fusion of cytotrophoblast cells, leading to the creation of a multinucleated syncytiotrophoblast–a tissue that coordinates the exchange of nutrients, waste metabolites and hormones between the fetus and the mother. The principal function of the human placenta is thus dependent on the fusogenic activity of Syncytin proteins.Our work focused on the development of a reliable and sensitive assay for cell-cell fusion assessment. In previous studies on Syncytins, various methods were used to quantify fusogenic activity. To detect fusion events, these methods often relied on manual nuclei counting within fused cells or utilized the split GFP complementation system. However, these approaches demanded extensive image analysis, which was time-consuming and sometimes not very accurate. To address this, we have created a new assay based on the complementation of two luciferase fragments, which reconstitute into a functional enzyme after cell-cell fusion is induced. We show that our method can be used to explore the fusogenic features of several retroviral proteins, their interaction with receptors, and even inhibitors. A deeper understanding of these interactions could shed light on the mechanisms contributing to the development of syncytiotrophoblast-related diseases.
Trvalý link: https://hdl.handle.net/11104/0349541