This video shows T cells responding to antigens of differing affinities. The cells express the ERK Kinase Translocation Reporter and the OT-I transgenic T cell receptor allowing them to respond to ovalbumin derived antigens. The T cells are dropped onto our unique functionalized surfaces at the beginning of the video and can be seen exhibiting oscillatory ERK dynamics in response to antigen over the course of imaging (2 min imaging interval over 3.5 hrs). Deciphering function of these oscillations is the subject of ongoing research in the Regot Lab.
ERK Kinase Translocation Reporter and the OT-I transgenic T cell response to antigens of differing affinities
ERK activity dynamics in mouse skin
This video shows a nuclear mask of cells in the basal layer of the skin pseudocolored based on ERK activity. A mouse line expressing ERK KTR in all cells was imaged by our long term collaborators in the Greco lab (Yale University SoM). The video shows a spontaneous wave of ERK activity traveling through the basal layer of the skin. While the function of these waves has been subject of intense research, it appears to be directly related to controlling tissue homeostasis and maintaining barrier function as a collective.
Collective response to oncogenes in epithelial monolayers
This video shows how epithelial monolayers respond to oncogene expressing cells. The red cells contain an inducible oncogene (RasG12V) that is expressed during the movie. All cells express the ERK Kinase Translocation Reporter (ERK KTR shown in green). At the beginning of the time lapse (images taken every 5 minutes) most cells have inactive ERK (as shown by the biosensor being nuclear). During induction of the RasG12V by the red cells, ERK is activated in WT neighboring cells. This phenomenon occurs via activation of the matrix metalloprotease ADAM17 which in turn triggers release of the EGFR ligand AREG. For more details see Aikin et. al. 2020.
IRAK1 localization and NFkB nuclear translocation in live cells exposed to IL1b
Fibroblasts expressing IRAK1-GFP (cyan) and p65(NFkb)-mRuby2 (magenta) were treated with Interleukin 1 beta. The video shows rapid clustering of IRAK1 at the same time that NFkb translocates into the nucleus. We showed that IRAK1 clustering in response to stimulation renders cells tolerant to further stimulation (see DeFelice et al 2019). This video actually shows how cells that already have clustering of IRAK1 at the beginning of the video fail to respond to the stimuli (meaning NFkb does not go nuclear in those cells).
Natural Killer Cells patrolling a monolayer of epithelial cells
MCF10A cells were stained with Hoesch dye (blue) and images upon contact with NK92 cells stained with a red dye (red). In addition, the media contains a chemical dye to monitor apoptosis through activation of caspase 3 (green). The video shows how NK cells move around epithelial cells and selectively induce apoptosis of some epithelial cells.
Oncogene Induced ERK Signaling Dynamics
Dual Sensor MCF10A cell lines containing H2B-iRFP nuclear marker (red), ERK-mRuby2 localization reporter (yellow), and an ERK KTR-mCerulean3 representing kinase activity (cyan), and indicated inducible oncogene constructs were treated with doxycycline (2μg/ml) at time 0. Images were collected every five minutes for an hour before and 12 hours after induction. Scale bar = 100μm. See Aikin et al 2020 for more details. This video shows how expression of different oncogenic mutations differentially alters ERK localization and ERK activity. Interestingly only when ERK activity is sustained, the nuclear ERK localization can be observed.
Segmentation and Tracking in action!!
The following video shows NIH3T3 cells (mouse fibroblasts) expressing a nuclear marker in the green channel (H2B-EGFP), the NF-kB transcription factor in the red Channel (p65-DsRed) and a genetically encoded biosensor for JNK activity in the blue channel (JNK KTR-mCerulean3). Upon addition of Interleukin 1Beta, both NF-kB and JNK are activated by the same upstream mechanisms. This results in the nuclear translocation of NF-kB (red) and a cytoplasmic translocation of the JNK KTR (blue) while the nuclear marker H2B (green) stays always nuclear. The video will play 3 times:
- Three channels merged in RGB.
- Starts with the three merged channels, then shows just the nuclear channel (H2B-EGFP) in gray scale and how we transform it into segmented objects that we can track over time. Cool!! We typically use these objects to calculate nuclear intensity.
- Starts with the gray scale movie for the red channel (p65-DsRed) then we overlay the cytoplasmic ring objects that we will use to quantify cytoplasmic mean intensity. Finally it goes back to display the three merged channels. Enjoy!
JNK KTR is specific for JNK activity
In the following video, cells expressing JNK KTR (Kinase Translocation Reporter) are stressed with anisomycin. Such stress activates the Stress Activated Protein Kinase JNK which will phosphorylate the JNK KTR construct. The negative charge introduced to the construct by this phosphorylation inhibits nuclear import and enhances nuclear export resulting in a localization change that can be easily observed. The whole process takes about 10 minutes (images are taken every 5 minutes). In sumary, when the KTR signal is nuclear the kinase not active and when its in cytoplasm the kinase is active. After an hour of stimulation, we add a specific inhibitor of JNK activity. Accordingly, JNK is not active anymore, the JNK KTR construct is dephosphorylated and therefore the construct is relocated back to the nucleus.
ERK activity fluctuates under basal conditions
In this video mouse fibroblasts (NIH3T3) expressing ERK KTR are imaged every 6 minutes. Kinase Translocation Reporters or KTRs convert kinase activity into a localization change. Briefly, when the signal is nuclear the kinase is inactive and when its cytoplasmic the kinase is active. Therefore, by observing the ratio we can estimate ERK activity. Cells are growing in media supplemented with 1% serum, without any stimulation. As you can see, ERK activity displays heterogeneous fluctuations among cells under basal conditions.
Multiplexed monitoring of MAP Kinase signaling
In this video, mouse fibroblast cells (NIH3T3) express Kinase Translocation Reporters (KTRs) for the three MAP Kinases in three different colors: ERK KTR in green, JNK KTR in red and p38 KTR in blue. Kinase Translocation Reporters are engineered kinase substrates that change localization upon phosphorylation. Basically, when the fluorescence is nuclear the kinase is inactive and when the fluorescence is cytoplasmic the kinase is active. Cells are imaged every 12 minutes. About 6 hours after the start of the movie, we add anisomycin which induces translational stress and activates all three MAP kinases. After 2 hours a specific JNK inhibitor is added. By the end of the movie an inhibition of ERK fluctuations is observed.
Stress regulated gene expression in yeast
In this video, a strain of budding yeast (Saccharomyces cerevisiae) is expressing the MAP Kinase Hog1 fused to mCherry (red) and has a genomic integration of the STL1 osmoresponsive promoter driving the expression of a quadruple venus (green). Cells are kept in a microfluidic device and at the begining of the movie, a 0.4M solution of NaCl is flowed in. In this context, yeast cells activate the Stress Activated Protein Kinase Hog1 which is then re-localized to the nucleus to coordinate gene expression changes. One of the regulated promoters is STL1 and therefore, after some time, the quadruple venus is expressed.
Cell Cycle studies with the FUCCI system
In this video cells express the transcription factor SMAD2 in the green Channel and the FUCCI cell cycle indicators (see reference below) in the blue and red Channels. In this system, cells have blue nuclear signal (Cdt1-mCerulean3) in G1 and change to red (Geminin-mCherry) in late G1 to stay red for S, G2 and M phases. This allows us to quantify cell cycle progression at single cell level while measuring the activity of the transcription factor SMAD2. In this case, cells are growing under no stimulation, in 1% serum for 40 hours. Note cells transitioning from blue to red and how mitosis occurs in red cells to create 2 cells that will later be expressing the blue marker.
Reference: Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.
Sakaue-Sawano A et al. Cell. 132, 487-498 (2008)
NF-kB regulated gene expression
Similar to what happens in yeast, upon induction with the cytokine TNF, the transcription factor NF-kB translocates to the nucleus to induce gene expression. In this video, mouse fibrobalsts express p65DSred (a component of NF-kB labeled in red) and also contain a synthetic promoter with multiple binding sites for NF-kB driving the expression of VenusFP (green). At the begining of the movie cells are stimulated with TNF which induces the translocation of NF-kB to the nucleus and after some time Venus is expressed in the green Channel.
NF-kB and JNK signaling simultaneously in live single cells
The following video shows a polyclonal line of NIH3T3 cells (mouse fibroblasts) expressing a nuclear marker in cyan (H2B-mCerulean3), the NF-kB transcription factor in the green Channel (p65-mClover) and a genetically encoded biosensor for JNK activity in the red Channel (JNK KTR-mRuby2). Upon addition of Interleukin 1Beta, both NF-kB and JNK are activated by the same upstream mechanisms. This results in the nuclear translocation of NF-kB (green) and a cytoplasmic translocation of the JNK KTR (red) while the nuclear marker H2B (blue) stays always nuclear.