1. Treadmill Running for Revealing the Extent of Muscle Injury in Mouse Models 
<ol>
 <li>Establish the angle of the treadmill surface for use during the running protocol. Generally, a flat level or an angle between 7&deg;and 15&deg;degrees downhill or uphill is used. Some treadmills have in integral apparatus to adjust the incline while others require the treadmill to be elevated through other means.</li>
 <li>Place a tray or a blue lab pad underneath the treadmill before putting the animals in the treadmill to collect any waste from the animals during the running protocol.</li>
 <li>Before running mice should be acclimated to the environment of the treadmill. This involves placing the animals in the treadmill for 15 minutes with the electric grid off and the belt drive motor on but the belt not moving (i.e. with the speed set at 0 m/s).</li>
 <li>Turn on the motivational electric grid. The intensity and frequency of the pulses used can usually be controlled on treadmills but varies from one manufacturer to another. Generally, maximum intensity is not required to motivate mice to run while a high frequency (at least once every two seconds) will improve compliance.</li>
 <li>Activate the treadmill belt to begin the running protocol. An initial speed of approximately 5 meters/min should be used for a warm up period for the mice. The speed of the treadmill can be accelerated slowly, usually by adding 1-2 meters/min for each minute following the start of the treadmill.</li>
 <li>Compliance of animals in most protocols can be improved by conducting a series of short runs (5 minutes) at warm up speed for 3 to 10 days before the experimental running begins.</li>
 <li>An acute exhaustion exercise will usually involve increasing the speed of the treadmill with time until a maximum speed (generally &lt; 30 m/m) is achieved and maintained until the mice show signs of exhaustion. Criteria for judging exhaustion vary but generally include a mouse spending more than half of the time on the electric grid or 5 consecutive seconds on the electric grid without returning to the treadmill surface. After an individual mouse is exhausted it can be removed from the treadmill and the total time of running can be recorded.</li>
 <li>Changing the angle of the treadmill can be used in acute running trials to increase the exercise load (with an uphill angle) or to induce damaging eccentric contractions (with a downhill angle) that tear the sarcolemmal membranes of muscle fibers1. Evans blue dye can be injected into animals before such procedures for use as a histological dye to identify damaged muscle fibers2,3.</li>
 <li>Endurance training running protocols provide an exercise load to the animal over a prolonged period of time (up to months) in order to produce remodeling of the muscle or the cardiovascular system. Warm up procedure is the same as above (step 1.5) however the maximum speed is generally lower and the running times can be quite long (&gt;1 hour).</li>
 <li>After completion of running the mice can be returned to their cages. Treadmills usually become quite dirty while the mice are running, particularly over long protocols. It is necessary to clean the treadmill, usually with a 70% ethanol spray, following each use.</li>
</ol>
2. Ex Vivo Assay of Membrane Repair Capacity of Isolated Muscle Fibers Following UV-laser Damage
<ol>
 <li>Dissect out the superficial layer of the flexor digitorum brevis (FDB) muscle from the mouse foot. First, cut the skin of the sole open at the median line while taking care not to damage the muscle underneath, then make horizontal cuts and remove the skin. The flat white tendon of FDB muscle (proximal tendon) can be seen attached to the calcaneus bone. Bluntly separate the tendon by forceps and sever the tendon close to site of attachment, grab the free tendon by forceps and pull it up gently to separate the FDB from surrounding tissues while cutting any connective tissues that remain attached. Upon seeing the distal tendons branch to the individual digits the FDB may be removed by cutting where these tendons connect to the deeper layer of muscle.</li>
 <li>Put the FDB muscle bundle in 1 ml of collagenase solution pre-warmed to 37&deg;C in a 1.5 ml Eppendorf tube, tape the tube in a horizontal orientation in a 37&deg;C orbital shaker and shake at 200 rpm for 65 min. Times of incubation may have to be adjusted to ensure sufficient digestion, which is indicated by a frayed appearance and pale color of the muscle.</li>
 <li>Carefully transfer the digested FDB bundle into a 1.5 &mu;l centrifuge tube containing ~600 &mu;l of 2.5 Ca2+ Tyrode via a 1 ml pipette tip with a cut end that has a diameter sufficient to allow the digested bundle to easily pass through without disruption. Gently flip the tube to shake the loose fibers from the bundle.</li>
 <li>Cut a 30 &mu;L pipette tip so that the diameter is only slightly smaller than the muscle bundle (between 15 - 20 &mu;m in diameter).Use the pipettor to gently draw the bundle into the pipette and then push it back into the solution. Repeat this process until the majority of the fibers are disassociated from the bundle.</li>
 <li>Mix disassociated fibers well by gently turning the tube, take desired amount and drop onto a glass-bottom delta T dish. The volume to load will vary based on the efficiency of isolation and the number of useful fibers that are needed on a dish for a particular protocol. The remaining fibers can be stored in the tube at 4&deg;C for approximately 6 hours for additional studies.</li>
 <li>Allow the dish to sit undisturbed for 5 minutes. This allows the fibers to adhere to the glass bottom of the dish.</li>
 <li>Place the glass bottomed dish on a confocal microscopy equipped with a UV laser. Observe the fibers under phase microscopy at &gt;100X magnification to check for the presence of a normal striation pattern and a straight, rod like shape.</li>
 <li>Add FM1-43 or FM4-64 styrylpyridinium dyes4to solution to a final concentration of 2.5 &mu;M5.</li>
 <li>To induce damage to the fiber position it in the imaging window so the fiber is oriented at a 45&deg; angle from the top left of the field of view to the bottom right (Figure 1). Irradiate a 5x5 pixel area (approximately 0.9&mu;mx0.9&mu;m)of the plasma membrane using a UV laser (80 mW or greater, 351/364 nm) set to maximum power for 5 seconds. The irradiated region should split the plasma membrane; that is, half of the 5x5 box should be inside the fiber, while the remainder should be outside the fiber (Figure 1).</li>
 <li>Capture images of dye entry into the fiber at intervals of 5 seconds each for up to 5 minutes.</li>
 <li>Repeat step 2.8 for no more than 3 fibers per dish, as the fluorescent dye will eventually be endocytosed into the fiber and complicate any data analysis. After 3 fibers a new dish should be prepared from step 2.5.</li>
 <li>Analyze data by calculating the change in fluorescence intensity (&Delta;F/F0) between each captured frame to measure the amount of dye entry. This requires measuring the mean fluorescence of an area approximately 200 &mu;m2 using analysis software such as the publically available ImageJ (http://rsbweb.nih.gov/ij/). For comparison between fibers, the following calculation should be made for each frame: &Delta;F/F0, where F0 is the mean fluorescence of the region of interest in the first captured frame (t = 0; prior to injury), and &Delta;F is the change in fluorescence of each subsequent frame (F-F0).</li>
</ol>
3. Live Cell Confocal Imaging to Monitor the Dynamic Process of Cell Membrane Repair
<ol>
 <li>Micropipettes were made from PYREX Capillaries. The capillary was placed on a micropipette puller and pulled by pre-set program (Heat=695; Pull=50; Vel.=55; Time=250).</li>
 <li>Micropipette was attached to a 3 axis (xyz) micromanipulator.</li>
 <li>Cells are transfected with plasmid DNA using normal methods on a glass bottom delta T dish. Media should be switched to 2.5mM Ca2+ Tyrode buffer before experiments begin.</li>
 <li>Glass dishes containing transfected cells were placed on a laser scanning confocal microscope with 40X 1.3NA oil immersion objective.</li>
 <li>The acute live-cell damage was performed by inserting the micropipette into the cell membrane and then quickly retract the micropipette out of the cell3,6. Consecutive live cell images were obtained at an interval of 1.54 s/frame.</li>
 <li>For the saponin induced membrane disruption assay, 0.005% saponin (Sigma) in 2.5mM Ca2+ Tyrode buffer was perfused by a gravity-flow custom-assembled perfusion system7. The perfusion rate is approximately 1 ml/min and the perfusion tip should be placed directly above the cell being imaged.</li>
</ol>
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.
<img src="/files/ftp_upload/2717/2717fig1.jpg" alt="Figure 1"/> 
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.
<img src="/files/ftp_upload/2717/2717fig2.jpg" alt="Figure 2"/> 
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.
<img src="/files/ftp_upload/2717/2717fig3.jpg" alt="Figure 3"/> 
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.

<ol>
	<li>Armstrong, R. B., Ogilvie, R. W., Schwane, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eccentric+exercise-induced+injury+to+rat+skeletal+muscle.">Eccentric exercise-induced injury to rat skeletal muscle.</a> J Appl Physiol. 54, 80-93 (1983).</li><li>Hamer, P. W., McGeachie, J. M., Davies, M. J., Grounds, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evans+Blue+Dye+as+an+in+vivo+marker+of+myofibre+damage:+optimising+parameters+for+detecting+initial+myofibre+membrane+permeability.">Evans Blue Dye as an in vivo marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability.</a> J Anat. 200, 69-79 (2002).</li><li>Cai, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MG53+nucleates+assembly+of+cell+membrane+repair+machinery.">MG53 nucleates assembly of cell membrane repair machinery.</a> Nat Cell Biol. 11, 56-64 (2009).</li><li>Cochilla, A. J., Angleson, J. K., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+secretory+membrane+with+FM1-43+fluorescence.">Monitoring secretory membrane with FM1-43 fluorescence.</a> Annu Rev Neurosci. 22, 1-10 (1999).</li><li>McNeil, P. L., Miyake, K., Vogel, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endomembrane+requirement+for+cell+surface+repair.">The endomembrane requirement for cell surface repair.</a> Proc Natl Acad Sci U S A. 100, 4592-4597 (2003).</li><li>Cai, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+repair+defects+in+muscular+dystrophy+are+linked+to+altered+interaction+between+MG53,+caveolin-3,+and+dysferlin.">Membrane repair defects in muscular dystrophy are linked to altered interaction between MG53, caveolin-3, and dysferlin.</a> J Biol Chem. 284, 15894-15902 (2009).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioprotection+of+ischemia/reperfusion+injury+by+cholesterol-dependent+MG53-mediated+membrane+repair.">Cardioprotection of ischemia/reperfusion injury by cholesterol-dependent MG53-mediated membrane repair.</a> Circ Res. 107, 76-83 (2010).</li><li>Knab, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeatability+of+exercise+behaviors+in+mice.">Repeatability of exercise behaviors in mice.</a> Physiol Behav. 98, 433-440 (2009).</li><li>Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+resistance+to+fatigue+and+altered+calcium+handling+properties+of+sarcalumenin+knockout+mice.">Enhanced resistance to fatigue and altered calcium handling properties of sarcalumenin knockout mice.</a> Physiol Genomics. 23, 72-78 (2005).</li><li>Massett, M. P., Berk, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-dependent+differences+in+responses+to+exercise+training+in+inbred+and+hybrid+mice.">Strain-dependent differences in responses to exercise training in inbred and hybrid mice.</a> Am J Physiol Regul Integr Comp Physiol. 288, 1006-1013 (2005).</li><li>Bansal, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+membrane+repair+in+dysferlin-deficient+muscular+dystrophy.">Defective membrane repair in dysferlin-deficient muscular dystrophy.</a> Nature. 423, 168-172 (2003).</li></ol>

No conflicts of interest declared.

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 &Delta;F/F0 is very sensitive to the region of interest selected for measurement of mean fluorescence. An area of approximately 200&mu;m2, 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 &Delta;F/F0 in fibers with little dye entry while lowering &Delta;F/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.

<table><tbody><tr><td>Rodent treadmill</td><td>Columbus Instruments Exer 3/6 or equivalent</td><td></td><td></td></tr><tr><td>70 % ethanol</td><td>ISC Bioexpress</td><td></td><td></td></tr><tr><td>Dissection tools including fine tip forceps, spring scissors</td><td>World Precision Instruments, Inc.</td><td></td><td></td></tr><tr><td>2 mg/ml collagenase type I</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>0 Ca&lt;sup&gt;2+&lt;/sup&gt; Tyrode buffer (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, pH 7.2, 290 mosm)</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>2.5mM Ca&lt;sup&gt;2+&lt;/sup&gt; Tyrode buffer (140 mM NaCl, 2.5 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 5 mM KCl, 10 mM HEPES, 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, pH 7.2, 290 mosm)</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Temperature controllable orbital shaker</td><td>New Brunswick Scientific</td><td></td><td></td></tr><tr><td>Delta TPG Dish</td><td>Fisher Scientific</td><td>1207133</td><td></td></tr><tr><td>LSM 510 confocal microscope with an Enterprise 80 mW UV laser or equivalent confocal microscope</td><td>Carl Zeiss, Inc.</td><td></td><td></td></tr><tr><td>FM1-43 or FM4-64 dyes</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Borosilicate Capillaries Size 0.8-1.0 X 100 mm</td><td>PYREX</td><td>Part No. 9530-2</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument Co.</td><td>model P-97</td><td></td></tr><tr><td>3 axis micromanipulator</td><td>Narishige International</td><td>MHW-3</td><td></td></tr><tr><td>Radiance 2100 laser scanning confocal microscope or equivalent microscope</td><td>Bio-Rad</td><td>MHW-3</td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr></tbody></table>

visualization of mg53-mediated cell membrane repair using in vivo and in vitro systems

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.

Watch this Scientific Journal Video about Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems at JoVE.com

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

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.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

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 ...

visualization-mg53-mediated-cell-membrane-repair-using-vivo-vitro

Nanyang Technological University

Research

JoVE Journal

Biology

12.3K Views.  Robert Wood Johnson Medical School. 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.

Video: Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Cancer Research

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...

Immunology and Infection

Murine Flexor Tendon Injury and Repair Surgery

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers and general hand function. Injuries to the FTs are common, and satisfactory healing is often impaired due to excess scar tissue and adhesions between the tendon and surrounding tissue. However, little is known about the molecular and cellular components of FT repair. To that end, a murine model of FT repair that recapitulates many aspects of healing in humans, including impaired range of motion and decreased mechanical properties, has been developed and previously described. Here an in-depth demonstration of this surgical procedure is provided, involving transection and subsequent repair of the flexor digitorum longus (FDL) tendon in the murine hind paw.&#160;This technique can be used to conduct lineage analysis of different cell types, assess the effects of gene gain or loss-of-function, and to test the efficacy of pharmacological interventions in the healing process. However, there are two primary limitations to this model: i) the FDL tendon in the mid-portion of the murine hind paw, where the transection and repair occur, is not surrounded by a synovial sheath. Therefore this model does not account for the potential contribution of the sheath to the scar formation process. ii) To protect the integrity of the repair site, the FT is released at the myotendinous junction, decreasing the mechanical forces of the tendon, likely contributing to increased scar formation. Isolation of sufficient cells from the granulation tissue of the FT during the healing process for flow cytometric analysis has proved challenging; cytology centrifugation to concentrate these cells is an alternate method used, and allows for generation of cell preparations on which immunofluorescent labeling can be performed. With this method, quantification of cells or proteins of interest during FT healing becomes possible.

Flexor tendons in the hand are commonly injured, leading to impaired hand function. However, the scar-tissue healing response is not well characterized. A murine model of flexor tendon healing is demonstrated here. This model can enhance overall understanding of the healing process and assess therapeutic approaches to improve healing.

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers ...

Medicine

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...

Laser Induced Thermoplasmonic Puncture for the Study of Plasma Membrane Repair

A Thermoplasmonic Approach for Investigating Plasma Membrane Repair in Living Cells and Model Membranes

The cell membrane is crucial for cell survival, and ensuring its integrity is essential as the cell experiences injuries throughout its entire life cycle. To prevent damage to the membrane, cells have developed efficient plasma membrane repair mechanisms. These&#160;repair mechanisms can be studied by combining confocal microscopy and nanoscale thermoplasmonics to identify and investigate the role of key proteins, such as annexins, involved in surface repair in living cells and membrane model systems.
The puncturing method employs a laser to induce highly localized heating upon nanoparticle irradiation. The use of near-infrared light minimizes phototoxicity in the biological sample, while the majority of the absorption takes place in the near-infrared resonant plasmonic nanoparticle. This thermoplasmonic method has been exploited for potential photothermal and biophysical research to enhance the understanding of intracellular mechanisms and cellular responses through vesicle and cell fusion studies. The approach has shown to be complementary to existing methods for membrane disruption, such as mechanically, chemically, or optically induced injuries, and provides a high level of control by inflicting extremely localized injuries. The extent of the injury is limited to the vicinity of the spherical nanoparticle, and no detrimental damage occurs along the beam path as opposed to pulsed lasers using different wavelengths. Despite certain limitations, such as the formation of nanobubbles, the thermoplasmonic method offers a unique tool for investigating cellular responses in plasma membrane repair in an almost native environment without compromising cell viability.
When integrated with confocal microscopy, the puncturing method can provide a mechanistic understanding of membrane dynamics in model membrane systems as well as quantitative information on protein responses to membrane damage, including protein recruitment and their biophysical function. Overall, the application of this method to reduced model systems can enhance our understanding of the intricate plasma membrane repair machinery in living cells.

Author Spotlight: Exploring Plasma Membrane Repair Mechanisms with Innovative Thermoplasmonic Puncturing 

The thermoplasmonic puncture method integrates confocal microscopy, optical tweezers, and gold nanoparticles to study protein responses during plasma membrane repair in cells and giant unilamellar vesicles. The technique enables rapid and localized membrane puncture, allowing the identification of key proteins and their functional roles in the intricate plasma membrane repair machinery.

The cell membrane is crucial for cell survival, and ensuring its integrity is essential as the cell experiences injuries throughout its entire life cycle. ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Summary

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Chapters in this video

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Murine Flexor Tendon Injury and Repair Surgery

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

Live Imaging Assay for Assessing the Roles of Ca²⁺ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

A Thermoplasmonic Approach for Investigating Plasma Membrane Repair in Living Cells and Model Membranes