About the Seminar:

Observing protein interactions in live cells has remained challenging due to their heterogeneous complexities. Methods such as Fluorescence Resonance Energy Transfer (FRET) have been used to track protein interactions but are limited to an energy transfer distance between molecules. Ephrin type-A Receptor 2 (EphA2) is a transmembrane protein that has two functions: tumor suppression and tumor signaling depending on the presence of a ligand. Previous studies that have attempted to characterize these interactions using FRET have provided conflicting reports about the assembly of the EphA2 assembly. The paper I will focus on (Xiaojun Shi et al., Science 382,1042-1050, 2023) uses a technique called pulse interleaved excitation-fluorescence cross-correlation spectroscopy (PIE-FCCS) to characterize EphA2 protein interactions in cells. PIE-FCCS provides a multiplex readout that characterizes oligomerization, mobility, density, and proximity. FCCS has been used to investigate other protein-protein interactions; the issue arises when the probe molecules undergo a process called crosstalk. This results in false positive signals being detected that are not accurate to the actual protein-protein interactions. PIE can be used to eliminate this crosstalk by using two different excitation sources to assign photon arrival. This seminar will focus on PIE-FCCS as a tool to characterize the ligand-free EphA2 assembly in cells which is less understood.