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. Cells are imaged every 12 minutes. About 6 hours later 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.
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 clonal line of NIH3T3 cells (mouse fibroblasts) expressing a nuclear marker in the green Channel (H2B-GFP), 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.
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.
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 (Cdt1-mCerulean3) nuclear signal 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)
DOCs: Dynamic Opto Cell stimulator
Light sensitive molecular switches have emerged as a powerful tool to interrogate signaling networks and their dynamic behavior. However, to address such questions light needs to be delivered with specific dynamic and spatial patterns. Such patterns can be easily achieved under the microscope by using a wide variety of solutions such as digital mirrors. These devices provide unique single cell (and subcellular) resolution but they are not appropriate for genome wide or proteomic approaches. Here we show our DOCs device able to stimulate cell populations in different growing formats (6 well plate, dishes, flasks…). In this video we show how we can regulate the amplitude of the signal in each well and then how we can generate oscillatory inputs of different frequencies. Learn more here!!
And don’t miss our interpretation of the Tetris song using DOCs 😀