real-time-monitoring-aurora-kinase-activation-using-conformational

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon ...

Univ Rennes, CNRS, IGDR (Genetics and Development Institute of Rennes), UMR 6290, F-35000 Rennes, France

New imaging methodologies in quantitative fluorescence microscopy and nanoscopy have been developed in the last few years and are beginning to be extensively applied to biological problems, such as the localization and quantification of protein interactions. Fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) is currently employed not only in biophysics or chemistry but also in bio-medicine, thanks to new advancements in technology and also new developments in data treatment. FRET-FLIM can be a very useful tool to ascertain protein interactions occurring in single living cells. In this review, we stress the importance of increasing the acquisition speed when working in vivo employing Time-Domain FLIM. The development of the new mathematical-based non-fitting methods allows the determining of the fraction of interacting donor without the requirement of high count statistics, and thus allows the performing of high speed acquisitions in FRET-FLIM to still be quantitative.

Non fitting based FRET-FLIM analysis approaches applied to quantify protein-protein interactions in live cells.

AURKA is a serine/threonine kinase overexpressed in several cancers. Originally identified as a protein with multifaceted roles during mitosis, improvements in quantitative microscopy uncovered several non-mitotic roles as well. In physiological conditions, AURKA regulates cilia disassembly, neurite extension, cell motility, DNA replication and senescence programs. In cancer-like contexts, AURKA actively promotes DNA repair, it acts as a transcription factor, promotes cell migration and invasion, and it localises at mitochondria to regulate mitochondrial dynamics and ATP production. Here we review the non-mitotic roles of AURKA, and its partners outside of cell division. In addition, we give an insight into how structural data and quantitative fluorescence microscopy allowed to understand AURKA activation and its interaction with new substrates, highlighting future developments in fluorescence microscopy needed to better understand AURKA functions in vivo. Last, we will recapitulate the most significant AURKA inhibitors currently in clinical trials, and we will explore how the non-mitotic roles of the kinase may provide new insights to ameliorate current pharmacological strategies against AURKA overexpression.

Insights into the non-mitotic functions of Aurora kinase A: more than just cell division.

Hepatitis B virus (HBV) core protein (HBc) is essential to the formation of the HBV capsid. HBc contains two domains: the N-terminal domain corresponding to residues 1-140 essential to form the icosahedral shell and the C-terminal domain corresponding to a basic and phosphorylated peptide, and required for DNA replication. The role of these two domains for HBV capsid assembly was essentially studied in vitro with HBc purified from mammalian or non-mammalian cell lysates, but their respective role in living cells remains to be clarified. We therefore investigated the assembly of the HBV capsid in Huh7 cells by combining fluorescence lifetime imaging microscopy/Förster's resonance energy transfer, fluorescence correlation spectroscopy and transmission electron microscopy approaches. We found that wild-type HBc forms oligomers early after transfection and at a sub-micromolar concentration. These oligomers are homogeneously diffused throughout the cell. We quantified a stoichiometry ranging from ~170 to ~230 HBc proteins per oligomer, consistent with the visualization of eGFP-containingHBV capsid shaped as native capsid particles by transmission electron microscopy. In contrast, no assembly was observed when HBc-N-terminal domain was expressed. This highlights the essential role of the C-terminal domain to form capsid in mammalian cells. Deletion of either the third helix or of the 124-135 residues of HBc had a dramatic impact on the assembly of the HBV capsid, inducing the formation of mis-assembled oligomers and monomers, respectively. This study shows that our approach using fluorescent derivatives of HBc is an innovative method to investigate HBV capsid formation.

Marc Tramier

Univ Rennes, CNRS, IGDR (Genetics and Development Institute of Rennes), UMR 6290, F-35000 Rennes, France

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells