KIT | KIT-Bibliothek | Impressum | Datenschutz

Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy

Eckert, A. F. 1; Gao, P. 1,2; Wesslowski, J. 3; Wang, X. 3; Rath, J. 1; Nienhaus, K. 1; Davidson, G. 3; Nienhaus, G. U. ORCID iD icon 1,2,3
1 Institut für Angewandte Physik (APH), Karlsruher Institut für Technologie (KIT)
2 Institut für Nanotechnologie (INT), Karlsruher Institut für Technologie (KIT)
3 Institut für Biologische und Chemische Systeme (IBCS), Karlsruher Institut für Technologie (KIT)

Abstract (englisch):

Development and homeostasis of multicellular organisms is largely controlled by complex cell-cell signaling networks that rely on specific binding of secreted ligands to cell surface receptors. The Wnt signaling network, as an example, involves multiple ligands and receptors to elicit specific cellular responses. To understand the mechanisms of such a network, ligand-receptor interactions should be characterized quantitatively, ideally in live cells or tissues. Such measurements are possible using fluorescence microscopy yet challenging due to sample movement, low signal-to-background ratio and photobleaching. Here, we present a robust approach based on fluorescence correlation spectroscopy with ultra-high speed axial line scanning, yielding precise equilibrium dissociation coefficients of interactions in the Wnt signaling pathway. Using CRISPR/Cas9 editing to endogenously tag receptors with fluorescent proteins, we demonstrate that the method delivers precise results even with low, near-native amounts of receptors.

Verlagsausgabe §
DOI: 10.5445/IR/1000120991
Veröffentlicht am 07.07.2020
DOI: 10.7554/eLife.55286
Zitationen: 21
Zitationen: 29
Cover der Publikation
Zugehörige Institution(en) am KIT Institut für Angewandte Physik (APH)
Institut für Biologische und Chemische Systeme (IBCS)
Institut für Nanotechnologie (INT)
Publikationstyp Zeitschriftenaufsatz
Publikationsjahr 2020
Sprache Englisch
Identifikator ISSN: 2050-084X
KITopen-ID: 1000120991
HGF-Programm 43.23.01 (POF III, LK 01) Advanced Optical Lithography+Microscopy
Erschienen in eLife
Verlag eLife Sciences Publications
Band 9
Seiten Article No. e55286
Vorab online veröffentlicht am 11.06.2020
Nachgewiesen in Scopus
Web of Science
KIT – Die Forschungsuniversität in der Helmholtz-Gemeinschaft
KITopen Landing Page