Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane

Autor(en): Loechte, Sara
Waichman, Sharon
Beutel, Oliver
You, Changjiang 
Piehler, Jacob 
Stichwörter: ACTIVATION; Cell Biology; ERYTHROPOIETIN RECEPTOR; FLUORESCENCE CORRELATION SPECTROSCOPY; FRET MICROSCOPY; I INTERFERON-RECEPTOR; LIVING CELLS; MASS-SPECTROMETRY; MUTATIONAL ANALYSIS; PROTEIN-PROTEIN INTERACTIONS; SUPPORTED MEMBRANES
Erscheinungsdatum: 2014
Herausgeber: ROCKEFELLER UNIV PRESS
Journal: JOURNAL OF CELL BIOLOGY
Volumen: 207
Ausgabe: 3
Startseite: 407
Seitenende: 418
Zusammenfassung: 
Interactions of proteins in the plasma membrane are notoriously challenging to study under physiological conditions. We report in this paper a generic approach for spatial organization of plasma membrane proteins into micropatterns as a tool for visualizing and quantifying interactions with extracellular, intracellular, and transmembrane proteins in live cells. Based on a protein-repellent poly(ethylene glycol) polymer brush, micropatterned surface functionalization with the HaloTag ligand for capturing HaloTag fusion proteins and RGD peptides promoting cell adhesion was devised. Efficient micropatterning of the type I interferon (IFN) receptor subunit IFNAR2 fused to the HaloTag was achieved, and highly specific IFN binding to the receptor was detected. The dynamics of this interaction could be quantified on the single molecule level, and IFN-induced receptor dimerization in micropatterns could be monitored. Assembly of active signaling complexes was confirmed by immunostaining of phosphorylated Janus family kinases, and the interaction dynamics of cytosolic effector proteins recruited to the receptor complex were unambiguously quantified by fluorescence recovery after photobleaching.
ISSN: 00219525
DOI: 10.1083/jcb.201406032

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