Fluorescence Lifetime Cross Correlation Spectroscopy Resolves EGFR and Antagonist Interaction in Live Cells

Fluorescence correlation or cross-correlation spectroscopy (FCS or FCCS), a single molecule technique, has the ability to provide highly sensitive information on interaction and dynamics of biomolecules both in vitro and in vivo. However, the inherent drawback of FCS is that species with similar molecular weight could not be differentiated. Although FCCS could resolve this through cross-correlation, it suffers from nonideal confocal volume overlap and spectral cross-talk which limits its application. In this work, we demonstrate for the first time the applicability of fluorescence lifetime correlation spectroscopy (FLCS) to monitor the interaction of an antagonist antibody with the epidermal growth factor receptor (EGFR) in live cells. As a proof of concept, we demonstrate the interaction of Cy5 labeled IgG and Alexa633 labeled anti-IgG using a single laser source (636 nm excitation) in vitro. The autocorrelation functions were separated based on their respective lifetime with a single detector and their K-d value was determined to be 11 +/- 3 nM. An in vivo application constituting the interaction of EGFR neutralizing antibody labeled with Alexa488 and EGFR-GFP in live 11EK293 cells was successfully demonstrated. The binding specificity of EGFR neutralizing antibody was confirmed by fluorescence lifetime cross-correlation measurements and fluorescence lifetime imaging (FLIM). The dissociation constant of this complex was found to be 9.2 +/- 2.7 nM. A quantitative assessment of receptor density calculations show that the density of EGFR significantly decreased, from 540 +/- 64 receptors/mu m(2) to 38 +/- 7 receptors/mu m(2) upon addition of the neutralizing EGFR antibody, indicating that the antagonist could induce receptor internalization. The demonstrated work not only opens up new opportunities in studying protein protein interactions in solutions and in live cells but also provide new insights in biology to understand how the antagonists influence EGFR through live cell quantification and imaging

Convergent evolution in the mechanisms of ACBD3 recruitment to picornavirus replication sites

Author summary Enteroviruses are the most common viruses infecting humans. They cause a broad spectrum of diseases ranging from common cold to life-threatening diseases, such as poliomyelitis. To date, no effective antiviral therapy for enteroviruses has been approved yet. To ensure efficient replication, enteroviruses hijack several host factors, recruit them to the sites of virus replication, and use their physiological functions for their own purposes. Here, we characterize the complexes composed of the host protein ACBD3 and the ACBD3-binding viral proteins (called 3A) of four representative enteroviruses. Our study reveals the atomic details of these complexes and identifies the amino acid residues important for the interaction. We found out that the 3A proteins of enteroviruses bind to the same regions of ACBD3 as the 3A proteins of kobuviruses, a distinct group of viruses that also rely on ACBD3, but are oriented in the opposite directions. This observation reveals a striking case of convergent evolutionary pathways that have evolved to allow enteroviruses and kobuviruses (which are two distinct groups of the Picornaviridae family) to recruit a common host target, ACBD3, and its downstream effectors to the sites of viral replication