Supplementary MaterialsVideo S1. To visualize ERK activity in thymocytes, we here developed knockin reporter mice expressing a F?rster/fluorescence resonance energy transfer (FRET)-based biosensor for ERK from your locus. Live imaging of thymocytes isolated from your reporter mice revealed that ERK regulates thymocyte motility in a subtype-specific manner. Unfavorable correlation between ERK activity and motility was observed in CD4/CD8 double-positive thymocytes and CD8 single-positive thymocytes, but not in CD4 single-positive thymocytes. Interestingly, however, the temporal deviations of ERK activity from the average correlate with the motility of CD4 single-positive thymocytes. Thus, live-cell FRET imaging will open a windows to understanding the dynamic nature and the diverse functions of ERK signaling in T?cell biology. locus. Live imaging of thymocytes has revealed that ERK activation suppresses thymocyte motility within the thymic microenvironment. Interestingly, we have revealed two different modes of CCNE2 translating ERK activity dynamics into cell motility in a manner dependent on cell types. The strength of ERK activity correlates negatively with cell motility in both the DP and CD8-SP subsets, whereas temporal deviations of ERK activity correlate with cell motility in the CD4-SP subset. These results suggest that cell motility of CD4-SP is usually more sensitive to ERK activity dynamics compared with the motility of other subsets under physiological conditions. Thus, the live-cell FRET imaging of ERK activity will open a windows to understanding the dynamic nature and the diverse functions of ERK signaling in T?cell biology. Results Lck-EKAREV-NLS Mice Enable ERK Activity Monitoring in T Cells EKAREV is usually a genetically encoded intramolecular FRET biosensor for monitoring ERK activity in living cells?(Physique?1A) (Komatsu et?al., 2011). EKAREV-NLS and EKAREV-NES contain a nuclear localization transmission?and a nuclear export transmission, respectively. In the first generation of transgenic mice, EKAREV was barely expressed in lymphocytes and gene silenced in some tissues. To express EKAREV ubiquitously, we launched the cDNAs of EKAREV-NLS and EKAREV-NES into the locus (Physique?1B) to?generate?knockin reporter mouse lines named Gt(ROSA)26Sortm1(CAG-loxP-tdKeima-loxP-EKAREV-NES) and?Gt(ROSA)26Sortm1(CAG-loxP-tdKeima-loxP-EKAREV-NLS) (hereinafter called R26R-EKAREV-NES and R26R-EKAREV-NLS), respectively. These mouse lines are designed to express the tdKeima fluorescent protein before Cre-mediated excision and EKAREV after excision, under the CAG promoter in the locus. Open in a separate window Physique?1 Lck-EKAREV-NLS Mice Enable ERK Activity Monitoring in Lymphocytes (A) A schema of EKAREV. Phosphorylation of the substrate peptide induces a conformational switch and a concomitant increase in the FRET efficiency. (B) A schema of the generation of R26R-EKAREV mice. Top to bottom: the structure of the targeting vector, the wild-type locus with the location of the insertion site, the structure of the sequence. Fragments shown in reddish and green can be expressed. The black AG-1478 cost rectangles around the left indicate the location of the first exon of the non-coding RNA in the locus. The gray rectangles indicate the location of the quit codons. sequences are indicated by black arrowheads. sequences are indicated by gray arrowheads. Neo is the neo cassette. DT-A is usually a diphtheria toxin A fragment gene for unfavorable selection. (C) Representative fluorescence images of EIIa-EKAREV-NES (left) and Eisuke (right) through a BA 520-560?nm filter shown in grayscale. The excitation wavelength was 840?nm. Top to bottom: the liver, the small intestine, and the lymph node. Left to right: image of EKAREV fluorescence and enlarged view of the left image. The yellow arrowheads show the regions with the promoter being inactive or only weakly active. Level bar, 30?m. (D) Circulation cytometric profile of EKAREV and CD3 expression among lymphocytes obtained from the lymph node of Lck-EKAREV-NLS. EKAREV expression is usually represented by YFP intensity. (E) Circulation cytometry of EKAREV-NLS expression in CD3+ AG-1478 cost lymphocytes of the lymph nodes derived from C57BL/6 (WT), Eisuke-NLS, and Lck-EKAREV-NLS mice. (F) Images of the paracortex region of the lymph node in a living mouse obtained by TPEM as AG-1478 cost shown in the schema. (Left) Fluorescence image of T?cells through a BA 520-560?nm emission filter. (Right) FRET/CFP ratio image shown in the intensity-modulated display (IMD) mode. Level bar, 20?m. (G) Representative FRET/CFP ratio images of the T?cells in the paracortex shown in IMD mode. Time-lapse imaging of T?cells in the paracortex is performed for 90?min. Anti-CD3? antibody (50?g/body) was injected intravenously at 0?min. After 60?min, MEK inhibitor (PD0325901) (100?g/body) was injected intravenously. The age of mouse in weeks is usually indicated. Left to right: FRET/CFP ratio image obtained just before anti-CD3? antibody administration, 60?min after anti-CD3? antibody administration, and 30?min after MEK inhibitor AG-1478 cost administration. Level bar, 25?m. (H) The FRET/CFP ratio just before anti-CD3? antibody administration (n?= 211 cells), 60?min after anti-CD3? antibody administration (n?= 212 cells), and 30?min after MEK inhibitor administration (n?= 207.