Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.
Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.
1. Treadmill Running for Revealing the Extent of Muscle Injury in Mouse Models
2. Ex Vivo Assay of Membrane Repair Capacity of Isolated Muscle Fibers Following UV-laser Damage
3. Live Cell Confocal Imaging to Monitor the Dynamic Process of Cell Membrane Repair
4. Representative Results:
A representative movie of the mouse treadmill running can be made during filming. The movie will illustrate the reduced running capacity of the mg53-/- mice due to damage to the skeletal muscle.
The extent of damage caused by the UV-laser damage protocol detailed above depends on the membrane repair capacity of the fiber analyzed. This capacity is affected by the genotype of the mouse from which the fiber was isolated, extracellular conditions, and any alterations to protein expression (transfection or infection). A normal wild type fiber will allow slight to moderate dye entry (Figure 2a). Muscle fiber derived from the mg53-/- mice with compromised membrane repair capacity will display more significant entry of dye into the fiber (Figure 2b).
Typical results for microelectrode injury show translocation of MG53-containing vesicles to the injury site. Figure 3a and 3b show GFP-MG53 transfected C2C12 myotubes damaged by insertion of the micropipette into the cell membrane. In the cell before micropipette damage, GFP-MG53 is located on both plasma membrane and cytoplasm3. Following micropipette penetration, GFP-MG53 containing vesicles moved towards damage sites (as indicated by arrowhead). Figure 3c shows a GFP-MG53 transfected C2C12 myoblasts treated with 0.005% of saponin, a detergent that can permeabilize the plasma membrane. Upon saponin treatment, GFP-MG53 translocated from cytosol to the cell membrane.
Weisleder, et al. Figure 1. Fiber alignment and injury region definition. Proper orientation of the isolated muscle fiber within the field of view and definition of the injury region shown.
Weisleder, et al. Figure 2. UV laser damage of FDB fiber. Images show fibers prior to injury (0 sec, left) and at 200 sec post injury (right). Fibers from a wild type mouse (a) show slight injury. Fibers from mice with compromised membrane repair (b) allow more dye to enter.
Weisleder, et al. Figure 3. Microelectrode and saponin damage to cultured cells. Images show C2C12 myotubes (a, b) and a C2C12 myoblast (c) prior to injury (above) and post injury (bottom). Cells injured with a microelectrode (a, b) show MG53 translocation to injury sites (arrows). Cells treated with saponin (c) show MG53 translocation to the cell membrane.
Treadmill running is a useful methodology at providing an exercise load to the treated animals. As a methodology for measuring muscle function or endurance capacity it is a notoriously noisy approach that is difficult to execute in a consistently reproducible fashion8. In general, times to exhaustion can be used as an endpoint to resolve major differences between experimental groups, such as those between some knockout mouse lines and wild type mice9. Experimental manipulations that produce more subtle difference, such as some pharmaceutical trials, may not resolve differences above the noise associated with this technique. In order to maximize the reproducibility and sensitivity of these techniques it is important to maintain the conditions for session to session very closely. The time of day the animals are exercised, as well as the warm up period conditions used, are important factors and should stay consistent from trial to trial.
Strain effects can have significant impact on the design of treadmill protocols as certain strains of mice can run for much more time on treadmill than other strains and respond differently to endurance exercise10. As a general guideline, many mice will be able to run 200-300 total meters in an exhaustion study with constant increase in the speed of the treadmill (5 m/m initial speed with 1 m/m added for each minute after start). For an endurance exercise the mice may be run between 9 to 12 m/m for 30 minutes 2 or 3 times a week. However, individual studies will usually require some optimization of the protocol to match individual experimental needs.
The UV-laser damage procedure has been shown to be an effective method for measuring the membrane repair capacity of skeletal muscle fibers3,6,11. One vital component to the success of these experiments is to use only muscle fibers that display a straight, rod shaped appearance with a regular pattern of striations and a smooth sarcolemma that does not appear wrinkled. If other muscle fibers are selected membrane damage may already be present in these fibers and interpretation of results from these experiments may be complicated. It is also important that the orientation of the fiber and definition of the irradiated region be as defined in Figure 1. This provides an injury of reproducible size, thus allowing comparison between fibers. If the defined region of injury lies completely within the fiber, an internal injury will be created rather than disrupting the sarcolemma. Additionally, the value calculated for ΔF/F0 is very sensitive to the region of interest selected for measurement of mean fluorescence. An area of approximately 200μm2, adjacent to and including the injury site, is appropriate. Unlike the region defined for injury, this area should lie completely within the fiber. If you include space outside the fiber, the resulting blank area will increase ΔF/F0 in fibers with little dye entry while lowering ΔF/F0 in fibers with a more severe phenotype. This decreases the sensitivity of the assay, thus making comparison between fibers more difficult.
For the micropipette damage assay, the angle between the micropipette and glass bottom dish should be approximately 45 degree in order to effectively damage the cell membrane. The cells were chosen for micropipette damage should be tightly attached to the bottom of the dish and rounded cells should not be used since most likely they will detach after micropipette poking. Saponin permeabilzation experiments are less technically demanding and relatively quicker to perform compared to micropipette penetration assays. However, there are several limitations to saponin damage, including that only one cell can be used per dish since saponin will diffuse and damage other cells in that dish.
The authors have nothing to disclose.
Name of the reagent | Company | Catalogue number |
---|---|---|
Rodent treadmill | Columbus Instruments Exer 3/6 or equivalent | |
70 % ethanol | ISC Bioexpress | |
Dissection tools including fine tip forceps, spring scissors | World Precision Instruments | |
2 mg/ml collagenase type I | Sigma | |
0 Ca2+ Tyrode buffer (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM MgCl2, pH 7.2, 290 mosm) | Sigma | |
2.5mM Ca2+ Tyrode buffer (140 mM NaCl, 2.5 mM CaCl2, 5 mM KCl, 10 mM HEPES, 2 mM MgCl2, pH 7.2, 290 mosm) | Sigma | |
Temperature controllable orbital shaker | New Brunswick or equivalent | |
Delta TPG Dish | Fisher Scientific | 1207133 |
LSM 510 confocal microscope with an Enterprise 80 mW UV laser or equivalent confocal microscope | Zeiss | |
FM1-43 or FM4-64 dyes | Invitrogen | |
Borosilicate Capillaries Size 0.8-1.0 X 100 mm | PYREX | Part No. 9530-2 |
Micropipette puller | Sutter Instruments | model P-97 |
3 axis micromanipulator | Narishige | MHW-3 |
Radiance 2100 laser scanning confocal microscope or equivalent microscope | BioRad | MHW-3 |
Saponin | Sigma | 47036 |