LifeAct Applied: Experimental Examples
- Restructuring of the Human Macrophage Cytoskeleton During Borreliae Uptake
- Super-Resolution Microscopy (STED) of the Actin Cytoskeleton
- Live Cell Imaging of Actin Dynamics in a Chemotactic Gradient
- Actin Dynamics Under Flow
- F-Actin Visualization in a 3D Hydrogel Matrix
- F-Actin Visualization in Living Cells Using the LifeAct-TagGFP2 Protein
- The Actin Cytoskeleton During Chemotaxis of Primary Murine T Cells
- Visualization of the Contraction of Cardiomyocytes
Borrelia bacteria are the cause of the Lyme disease, also known as Lyme borreliosis. To prevent the dissemination of borreliae, their uptake and elimination by macrophages has been shown to be necessary. This process involves dynamic restructuring of the macrophage cytoskeleton, particularly of the actin microfilaments.
In this experiment, the LifeAct Plasmid was used to visualize actin cytoskeleton reorganization in human macrophages during phagocytosis of borreliae.
Phagocytosis of borreliae by a primary human macrophage. Time-lapse movie of a confocal z-stack showing a primary human macrophage expressing RFP-LifeAct (red) internalizing several GFP-expressing spirochetes (green) with actin-rich cell protrusions. Sequence 41 min. Data by Dr. Mirko Himmel and Prof. Stefan Linder, PhD, Universitätsklinikum Hamburg-Eppendorf, Germany, http://www.linderlab.de/.
Using LifeAct-TagGFP2 Protein, the actin cytoskeleton can be visualized in detail. In this experiment, fixed Rat1 fibroblasts were incubated with LifeAct-TagGFP2 Protein in a µ-Slide VI 0.4, ibiTreat. Simulated emission depletion (STED) microscopy was performed to create a super-resolution image.
Super-resolution microscopy of the actin cytoskeleton in Rat1 fibroblasts using LifeAct-TagGFP2 Protein. Microscopy was performed on the STEDYCON super-resolution STED nanoscopy system (Abberior Instruments GmbH, Göttingen, Germany) with a Plan-Neofluar 100x/1.4 objective lens.
F-actin networks play an important role during cell migration, which can be investigated in detail using chemotactic gradients. Primary dendritic cells were isolated from mice and transfected with the LifeAct Plasmid.
For the chemotaxis assay, cells were seeded on the µ-Slide Chemotaxis and a chemotactic gradient (CCL19) was applied. One day after the transfection, F-actin dynamics in the migrating cells were visualized using live cell imaging.
Live cell imaging of actin dynamics in a LifeAct-expressing primary dendritic mouse cell after the application of a chemotactic gradient.
Several cell types in biofluidic vessels, such as endothelial cells and immune cells, are constantly exposed to shear stress in vivo. This mechanical stimulus has a great impact on the physiological behavior and adhesion properties of cells, and should be taken into account when performing respective studies.
By combining the ibidi channel slides, µ-Slide I Luer or µ-Slide VI 0.4, and the ibidi Pump System with ibidi’s LifeAct technology, the F-actin cytoskeleton can be visualized in living cells under shear stress conditions. The ibidi Pump System is ideal for the long-term application of physiological shear stress to a cell layer and enables the adjustment of different flow rates. The system is fully compatible with live cell imaging and high resolution fluorescence microscopy. Optionally, the fixation and immunofluorescence staining of the cells can be directly performed in the µ-Slide I Luer.
- Device: ibidi Pump System
- Slide: µ-Slide I 0.4 Luer (ibiTreat)
- Cells: LifeAct-expressing endothelial cells (HUVEC, P1), transduced with the LifeAct Adenoviral Vector rAV-LifeAct-TagGFP2
- Reagents: LifeAct Adenoviral Vector rAV-LifeAct-TagGFP2
- Shear stress parameters: 20 dyn/cm2
Live cell imaging under flow: actin cytoskeleton visualization in HUVEC after transduction with the LifeAct Adenoviral Vector rAV-LifeAct-TagGFP2 and cultivation under 20 dyn/cm2.
It is well known that cells behave differently in a 3D environment than in the conventional 2D cell culture. For F-actin visualization in migrating cells in a 3D culture system, Stably LifeAct-expressing HT-1080 cells were embedded in a synthetic hydrogel. The polymerized cell-hydrogel mixture was immobilized on a µ-Slide Angiogenesis . After 20 hours, Z-stacks of the whole cell body were collected using high resolution confocal microscopy. The Z-stacks were projected to merged images, accurately showing the F-actin dynamics of each single cell in a 3D matrix.
Z-stack of HT-1080 LifeAct-TagGFP2 cells in a 3D hydrogel environment.
F-Actin Visualization in Living Cells Using the LifeAct-TagGFP2 Protein
The LifeAct-TagGFP2 Protein is ideally suited for the quick and efficient visualization of the actin cytoskeleton in living cells. For the staining procedure, you can use any method for protein transfer that works for your cells of interest.
Rat1 fibroblasts were grown until confluency and washed with PBS before adding LifeAct-TagGFP2 Protein solution (30 µg/ml). Cells were scraped several times with a sterile pipette tip and incubated at 37°C for 5 minutes, leading to mechanical perturbation of the cell membrane and protein incorporation along the scrape. After a further washing step with PBS, medium was replaced and cells were imaged immediately.
Live cell imaging of F-actin in Rat1 fibroblasts after LifeAct-TagGFP2 Protein transfer (30 µg/ml, 3 minutes).
The Actin Cytoskeleton During Chemotaxis of Primary Murine T Cells
Primary T cells were isolated from the spleen of a LifeAct mouse. An under-agarose assay (UA-assay) was performed to analyze chemotaxis and chemokinesis. Fluorescent live cell images illustrate the movement of the LifeAct-stained actin cytoskeleton.
J. Riedl, K. C. Flynn, A. Raducanu, F. Gärtner, G. Beck, M. Bösl, … R. Wedlich-Söldner. Lifeact mice for studying F-actin dynamics. Nature Methods, 2010, 10.1038/nmeth0310-168
B. Heit and P. Kubes. Measuring Chemotaxis and Chemokinesis: The Under-Agarose Cell Migration Assay. Science Signaling, 2003, 10.1126/stke.2003.170.pl5
Live cell imaging of the actin cytoskeleton in migrating primary T cells.
For the visualization of the contraction rates of cardiomyocytes, Fuse-It-mRNA vesicles (Beniag) were filled with mRNA LifeAct-TagGFP2 and fused with human iPSC-derived cardiomyocytes. 16 hours after mRNA LifeAct-TagGFP2 transfer, the contractions per minute were measured. The myocytes showed contraction rates of about 70 beats per minute. This value is in the normal range of unmodified myocytes which show 50 to 80 contractions per minute.