Determination of Single Molecule erbB1 Homodimer Lifetimes Using Single Quantum Dot Tracking and a Diffusive Hidden Markov Model

Abstract

Signaling by the erbB family of transmembrane tyrosine kinase receptors controls many cellular pathways, including growth and differentiation, while dysregulation of erbB signaling is a hallmark of many cancers. To understand how receptor interactions regulate signaling, we have developed new multi-color single molecule tracking and analysis techniques. Diffusion and dimerization of erbB1 receptors on live cells were monitored with single particle tracking by using antibody fragments or ligand probes coupled to quantum dots (QDs). As a probe for resting receptors, we used a non-activating, non-competing, monovalent fragment of an anti-erbB1 heavy-chain only antibody (VhH). To track activated receptors, QDs conjugated to EGF ligand were employed. Dimer lifetimes were determined from two-color single particle trajectories using a modified Diffusive Hidden Markov Model. This analytic two-state (bound and free) model uses the two-color QD trajectories to find bound states and quantify the dimerization lifetime using a global estimation over many trajectories. We capture the behavior of erbB1 receptors diffusing on the surface of live A431 cells, including the formation of long-lived dimers between two EGF-QD-erbB1 complexes. Treatment with PD153035, a specific inhibitor of erbB1 tyrosine kinase activity, increases receptor diffusion rate and reduces the lifetime of erbB1 homodimers, suggesting a role for the active kinase domain in formation of stable dimers. These results demonstrate the capabilities of innovative imaging and analysis approaches to measure protein-protein interaction kinetics in real time.