A Microbead Supported Membrane-Based Fluorescence Imaging Assay Reveals Intermembrane Receptor-Ligand Complex Dimension with Nanometer Precision.
Receptor-ligand complexes spanning a cell-cell interface inevitably establish a preferred intermembrane spacing based on the molecular dimensions and orientation of the complexes. This couples molecular binding events to membrane mechanics and large-scale spatial organization of receptors on the cell surface. Here, we describe a straightforward, epi-fluorescence-based method to precisely determine intermembrane receptor-ligand dimension at adhesions established by receptor-ligand binding between apposed membranes in vitro. Adhesions were reconstituted between planar and silica microbead supported membranes via specific interaction between cognate receptor/ligand pairs (EphA2/EphrinA1 and E-cadherin/anti-E-cadherin antibody). Epi-fluorescence imaging of the ligand enrichment zone in the supported membrane beneath the adhering microbead, combined with a simple geometrical interpretation, proves sufficient to estimate intermembrane receptor-ligand dimension with better than 1 nm precision. An advantage of this assay is that no specialized equipment or imaging methods are required.
Mechanobiology Institute, National University of Singapore , Singapore 117411, Singapore.
Kabir H
KH
Biswas
Mechanobiology Institute, National University of Singapore , Singapore 117411, Singapore.|Department of Chemistry, University of California , Berkeley, California 94720, United States.
Jay T
JT
Groves
D000906:Antibodies / Q000737:chemistry*; D015703:Antigens, CD; D015820:Cadherins / Q000737:chemistry*; D036382:Ephrin-A1 / Q000737:chemistry*; D036383:Ephrin-A2 / Q000737:chemistry*; D006801:Humans; D008863:Microspheres; D061848:Optical Imaging; D036104:Receptor, EphA2; D012822:Silicon Dioxide / Q000737:chemistry*
D000906:Antibodies; D015703:Antigens, CD; C513384:CDH1 protein, human; D015820:Cadherins; C000623258:EPHA2 protein, human; D036382:Ephrin-A1; D036383:Ephrin-A2; D012822:Silicon Dioxide; D036104:Receptor, EphA2