Tethered Fluorogen Assay to Visualize Membrane Apposition in Living Cells

Bioconjug Chem. 2017 May 17;28(5):1356-1362. doi: 10.1021/acs.bioconjchem.7b00047. Epub 2017 Apr 26.

Abstract

We describe proof-of-concept for a novel approach for visualizing regions of close apposition between the surfaces of living cells. A membrane-anchored protein with high affinity for a chemical ligand is expressed on the surface of one set of cells, and the cells are co-cultured with a second set of cells that express a membrane-anchored fluorogen-activating protein (FAP). The co-cultured cells are incubated with a bivalent reagent composed of fluorogen linked to the high-affinity ligand, with the concentration of the bivalent reagent chosen to be less than the binding constant for the FAP-fluorogen pair but greater than the binding constant for the ligand-high-affinity protein pair. In these conditions, strong FAP signal is observed only in regions of close proximity between membranes of the two classes of cell, where high local concentration of fluorogen favors binding to the FAP.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / metabolism*
  • Biological Assay / methods*
  • Biosensing Techniques / methods*
  • Cell Membrane / metabolism*
  • Fluorescent Dyes / chemistry*
  • HEK293 Cells
  • Humans
  • Microscopy, Fluorescence
  • Protein Binding
  • Receptors, Platelet-Derived Growth Factor / metabolism*

Substances

  • Antibodies, Monoclonal
  • Fluorescent Dyes
  • Receptors, Platelet-Derived Growth Factor