Transcript Slide 1
Chemomechanical mapping of ligand-receptor binding kinetics on cells Sunyoung Lee, Jelena Mandic, and Krystyn J. Van Vliet PNAS, 2007 Dawn Spelke 20.309 November 20, 2008 Background • Binding of ligands to cell-surface receptors is critical for many cellular functions • Receptor location and kinetics of ligand binding are important • However, few experimental techniques allow the simultaneous study of both parameters • Application: clinical therapies (KD) Goal • Map receptor locations at the single molecule level • Measure binding kinetics at the single cell level • Experimental model: human umbilical vein endothelial cells (HUVECs) expressing vascular endothelial growth factor receptor 2 (VEGFR2) • Key target: mechanosensory function Experimental Design • Functionalized force imaging of chemically fixed and living HUVECs • Scanning probe microscopy • Molecular force spectroscopy • Cantilever probes functionalized with antiVEGFR2 antibody (~1 antibody/probe) anti-IgG1 labeled anti-IgG1 labeled antiVEGFR2 PEG linker Receptor Location • Scan surface: unbinding force between probe and surface retards full-amplitude oscillation • Competitive binding using soluble antiVEGFR2 demonstrates binding specificity → Conclude VEGFR2 • 1.47 ± 0.38x105 VEGFR2/cell: agrees! Phase Image No Competition Competitive Binding Receptor Location • Height images of topography • Four cytoskeletal bundles • Competitive binding does not degrade topography • Majority of receptors associated with underlying cytoskeletal bundles! No Competitive Competition Binding Binding Kinetics • Force-displacement profile (single ligandreceptor unbinding event) • Determine rupture force FR • Compare highrecognition and lowrecognition sites High Recognition • Calculate koff Low Recognition Binding Kinetics • Determine kon by imaging cell surface during competitive inhibition • Analyze temporal increase in bound receptor • Calculate KD Visualization on Living Cells • Detect unbinding events in same way • Time variation due to lateral diffusion of receptor and receptor recycling • However, minimal displacement relative to moving cytoskeleton! Time Lapse of 30 Minutes Significance and Future Work • Can answer questions about effect of colocalization of subcellular structures on receptor function and cell processes • Investigate other cell surface receptors/ molecules with this technique • Enable predictions of key dynamic interactions between extracellular molecules and cell surface Questions?