Live-Cell FRET Imaging Reveals a Role of ERK Activity Dynamics in Thymocyte Motility
Authors: Konishi Yoshinobu , Terai Kenta , Abe Takaya , Hamazaki Yoko , Takaori-Kondo Akifumi , Matsuda Michiyuki 
Published: October 2019

Abstract
Thymocyte motility is key to orchestrating migration between different thymic microenvironments at the appropriate developmental stage. Several lines of evidence suggest that extracellular signal-regulated kinase (ERK) signaling pathways plays critical roles in T cell development. Nevertheless, the dynamics of ERK activity and its role in regulating cell motility remain largely unknown due to technical difficulties. Therefore, there is an increasing demand for genetic reporter systems that provide the information of thymocyte motility and ERK signaling under physiological condition in real time.
To meet the demand, we developed transgenic mice expressing fluorescence resonance energy transfer (FRET) based biosensor for ERK incorporated into the ROSA26 locus conditionally. EKAREV is a genetically encoded intramolecular FRET biosensor for monitoring ERK activity in living cells. We introduced cDNAs of EKAREV into the ROSA26 locus to generate mouse lines named R26-EKAREV-NLSfl/+. In these mouse lines, EKAREV will be expressed in the nucleus after Cre-mediated excision of the loxP-flanked tdKeima-coding sequence. To study ERK activity dynamics in T cells, R26-EKAREV-NLSfl/+ were crossed with Lck-Cre mice to generate R26-EKAREV-NLSlck/+. R26-EKAREV-NLSlck/+ showed uniform and high-level expression of EKAREV in lymphocytes and allowed us to examine the ERK activity of T cells in vivo.
After initial characterization of R26-EKAREV-NLSlck/+, we attempted to unravel the potential crosstalk between ERK activity dynamics and cell motility within the thymic microenvironment. Long-term in vivo imaging of thymus was difficult due to the anatomical location abutting the heart. To circumvent this problem, thymocytes obtained from R26-EKAREV-NLSlck/+ were overlaid on C57BL/6 (WT) thymic slices and observed under two-photon excitation microscopy. During the course of experiments, we noticed a negative correlation between ERK activity and migration speed of thymocytes. To confirm this observation, thymocytes were cultured in the presence or absence of an mitogen-activated protein kinase/ERK kinase (MEK) inhibitor prior to transfer onto thymic slices. Treatment of thymocytes with MEK inhibitor increased averaged migration speed in both double-positive (DP) and single-positive (SP) subsets. Moreover, time-lapse imaging of each subsets of thymocytes revealed that the deviation of ERK activity from the average of individual cells regulated cell motility in CD4-SP, while the absolute value of ERK activity regulated cell motility in DP and CD8-SP. Collectively, these results suggest that CD4-SP is unique in that the ERK activity dynamics negatively regulate cell motility.
Which signal regulates ERK activity and cell motility of CD4-SP in the medulla? Interaction between T cell receptor (TCR) and major histocompatibility complex (MHC) is known to regulate ERK activity and cell motility of T cells, but their direct relationship remains unknown in tissues. To examine this possibility, CD4-SP thymocytes were overlaid onto WT or MHC class II knockout (KO) thymic slices. When overlaid onto KO thymic slices, the average migration speed was significantly increased, indicating that CD4-SP cells arrest upon TCR-MHC II interaction. Moreover, the variance of ERK activity, but not the average of it, in CD4-SP cells on KO thymic slices was decreased, suggesting that TCR-MHC II interaction contribute the variance of ERK activity in CD4-SP.
Our findings unravel that the deviation of ERK activity induced by TCR-MHC interactions negatively regulate cell motility of CD4-SP in the medulla. The live-cell FRET imaging of ERK activity will open a new window to understand the dynamic nature and the diverse functions of ERK signaling in T cell biology.

Disclosures
Takaori-Kondo: Janssen Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Celgene: Honoraria, Research Funding.