Single particle tracking-based reaction progress kinetic analysis reveals a series of molecular mechanisms of cetuximab-induced EGFR processes in a single living cell.

Cellular processes occur through the orchestration of multi-step molecular reactions. Reaction progress kinetic analysis (RPKA) can provide the mechanistic details to elucidate the multi-step molecular reactions. However, current tools have limited ability to simultaneously monitor dynamic variations in multiple complex states at the single molecule level to apply RPKA in living cells. In this research, a single particle tracking-based reaction progress kinetic analysis (sptRPKA) was developed to simultaneously determine the kinetics of multiple states of protein complexes in the membrane of a single living cell. The subpopulation ratios of different states were quantitatively (and statistically) reliably extracted from the diffusion coefficient distribution rapidly acquired by single particle tracking at constant and high density over a long period of time using super-resolution microscopy. Using sptRPKA, a series of molecular mechanisms of epidermal growth factor receptor (EGFR) cellular processing induced by cetuximab were investigated. By comprehensively measuring the rate constants and cooperativity of the molecular reactions involving four EGFR complex states, a previously unknown intermediate state was identified that represents the rate limiting step responsible for the selectivity of cetuximab-induced EGFR endocytosis to cancer cells.