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Dual-color dual-focus line-scanning FCS for quantitative analysis of receptor-ligand interactions in living specimens

Dörlich, R.M.; Chen, Q.; Hedde, P.N.; Schuster, V.; Hippler, M.; Wesslowski, J.; Davidson, G.; Nienhaus, G.U.

Cellular communication in multi-cellular organisms is mediated to a large extent by a multitude of cell-surface receptors that bind specific ligands. An in-depth understanding of cell signaling networks requires quantitative information on ligand-receptor interactions within living systems. In principle, fluorescence correlation spectroscopy (FCS) based methods can provide such data, but live-cell applications have proven extremely challenging. Here, we have developed an integrated dual-color dual-focus line-scanning fluorescence correlation spectroscopy (2c2f lsFCS) technique that greatly facilitates live-cell and tissue experiments. Absolute ligand and receptor concentrations and their diffusion coefficients within the cell membrane can be quantified without the need to perform additional calibration experiments. We also determine the concentration of ligands diffusing in the medium outside the cell within the same experiment by using a raster image correlation spectroscopy (RICS) based analysis. We have applied this robust technique to study the interactions of two Wnt antagonists, Dickkopf1 and Dickkopf2 (Dkk1/2), to their cognate receptor, low-density-lipoprotein-receptor related protein 6 (LRP6), in the plasma membrane of living HEK293T cells. ... mehr

Volltext §
DOI: 10.5445/IR/1000048018
DOI: 10.1038/srep10149
Zitationen: 19
Zitationen: 22
Cover der Publikation
Zugehörige Institution(en) am KIT Institut für Angewandte Physik (APH)
Institut für Nanotechnologie (INT)
Institut für Toxikologie und Genetik (ITG)
Publikationstyp Zeitschriftenaufsatz
Publikationsjahr 2015
Sprache Englisch
Identifikator ISSN: 2045-2322
KITopen-ID: 1000048018
HGF-Programm 47.01.01 (POF III, LK 01) Biol.Netzwerke u.Synth.Regulat. ITG+ITC
Erschienen in Scientific Reports
Verlag Nature Research
Band 5
Seiten 10149
Bemerkung zur Veröffentlichung Gefördert durch den KIT-Publikationsfonds
Schlagwörter Confocal microscopy, Biophysics
Nachgewiesen in Web of Science
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