ErbB family receptor dimerization dynamics and dysregulation via long-term single-molecule imaging.

Dimerization is crucial for the activation of ErbB family receptors, yet the real-time dynamics and effects of oncogenic mutations remain unclear. Here, we performed long-term, multicolor single-particle tracking (SPT) of EGFR, HER2, and HER3 in living cells using upconverting nanoparticles (UCNPs), which do not photobleach. Our technique enables continuous observation of receptor interactions, revealing details of their dimerization dynamics. Oncogenic EGFR mutations promote stable, ligand-independent dimerization. Unexpectedly, both HER2 and HER3 exhibit constitutive homodimerization, prompting a revised model for their activation mechanisms. HER2 mutations modestly enhance homodimer stability compared with EGFR mutations, while HER3 mutations destabilize homodimers, suggesting that HER3 homodimerization sequesters HER3 and limits heterodimerization with other receptors. We also identified stable, ligand-independent heterodimers among all three receptors, further stabilized by ligand stimulation. These insights offer a comprehensive ErbB interaction network, elucidating diverse dimerization mechanisms and implications for oncogenic signaling.