Real-time monitoring of cell surface protein arrival with split luciferases.
Split luciferases
apical membrane traffic
basolateral membrane traffic
cell polarization
epithelial cells
protein sorting
Journal
Traffic (Copenhagen, Denmark)
ISSN: 1600-0854
Titre abrégé: Traffic
Pays: England
ID NLM: 100939340
Informations de publication
Date de publication:
10 2023
10 2023
Historique:
revised:
20
06
2023
received:
27
02
2023
accepted:
22
06
2023
medline:
11
9
2023
pubmed:
5
7
2023
entrez:
5
7
2023
Statut:
ppublish
Résumé
Each cell in a multicellular organism permanently adjusts the concentration of its cell surface proteins. In particular, epithelial cells tightly control the number of carriers, transporters and cell adhesion proteins at their plasma membrane. However, sensitively measuring the cell surface concentration of a particular protein of interest in live cells and in real time represents a considerable challenge. Here, we introduce a novel approach based on split luciferases, which uses one luciferase fragment as a tag on the protein of interest and the second fragment as a supplement to the extracellular medium. Once the protein of interest arrives at the cell surface, the luciferase fragments complement and generate luminescence. We compared the performance of split Gaussia luciferase and split Nanoluciferase by using a system to synchronize biosynthetic trafficking with conditional aggregation domains. The best results were achieved with split Nanoluciferase, for which luminescence increased more than 6000-fold upon recombination. Furthermore, we showed that our approach can separately detect and quantify the arrival of membrane proteins at the apical and basolateral plasma membrane in single polarized epithelial cells by detecting the luminescence signals with a microscope, thus opening novel avenues for characterizing the variations in trafficking in individual epithelial cells.
Substances chimiques
Membrane Proteins
0
Luciferases
EC 1.13.12.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
453-462Informations de copyright
© 2023 The Authors. Traffic published by John Wiley & Sons Ltd.
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