Experimental Example: Restructuring of the Human Macrophage Cytoskeleton During Borreliae Uptake
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/.
LifeAct: A Versatile Tool for F-Actin Visualization in Living Cells
LifeAct, a 17-amino acid peptide, is derived from a protein from Saccharomyces cerevisiae. LifeAct stains filamentous actin (F-actin) structures in living or fixed eukaryotic cells and tissues. In contrast to GFP-actin and its alternatives such as actin-binding proteins, LifeAct does not interfere with actin dynamics in vitro and in vivo, as shown in several cell types and model organisms.*
LifeAct-Plasmids may be used in many cell types when a transient or stable expression of LifeAct is needed. In this case, researchers can choose between two different promoters, CMV (cytomegalovirus) or CAG (modified chicken ?-actin). For many standard cell lines, the CMV promoter is the best choice as it leads to strong expression of LifeAct. However, when working with sensitive cells such as neuronal or embryonic stem cells, it is most often recommended to use the CAG promoter, because it does not have a viral origin and an interference with cellular defense mechanisms ("silencing mechanisms") is less likely.
After transfection of cells with pLifeAct, F-actin is visualized using the fluorescence markers TagGFP2 or TagRFP**. Stable transfection of cell lines allows long-term actin staining for various applications.
Adenoviral vectors are available for difficult-to-transfect cells or when up to 100 % transgene expressing cells are needed. In this case, it is also possible to choose between the two promoters mentioned before.
In addition, different stable cell lines are available, which help facilitate daily lab life. You can immediately start performing experiments without the time-consuming and laborious generation of stable cell lines, while still assuring high-quality standards.
Method Comparison: Using LifeAct for F-Actin Visualization in Living Cells
Dr. Christiane Wiesner, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
“We use LifeAct-TagRFP and LifeAct-TagGFP2 plasmids in primary human macrophages. The LifeAct probes put us in the position to detect F-actin-rich adhesion structures, without the drawback of disturbing their dynamics. The signal is bright and clear without the background of nonintegrated G-actin. LifeAct gives us the opportunity to highlight F-actin-enriched, cytoskeletal organizations, without the disadvantages of fluorophore-tagged actin overexpression.”
Dr. Christiane Wiesner University Medical Center Hamburg-Eppendorf Hamburg Germany