Name: cell membrane repair assay using a two-photon laser microscope
Uploaded: 2018-01-02T15:00:00.000+00:00
Duration: 6 min 35 s
Description: Watch this Scientific Journal Video about Cell Membrane Repair Assay Using a Two-photon Laser Microscope at JoVE.com

This work was supported by University of Alberta Faculty of Medicine and Dentistry, The Friends of Garrett Cumming Research Chair Fund, HM Toupin Neurological Science Research Chair Fund, Muscular Dystrophy Canada, Canada Foundation for Innovation (CFI), Alberta Advanced Education and Technology (AET), Canadian Institutes of Health Research (CIHR), Jesse's Journey - The Foundation for Gene and Cell Therapy, the Women and Children's Health Research Institute (WCHRI), and Alberta Innovates Health Solutions (AIHS).
We would like to thank Dr. Steven Laval for supplying us with the full-length dysferlin plasmid. We would also like to thank Dr. Katsuya Miyake for technical advice.

Human fibroblast cells used in this study were used with approval from the Human Ethics Committee of the Faculty of Medicine and Dentistry at the University of Alberta.
1. Transfection of cells with full-length DYSF plasmid
<ol>
	<li>Culture fibroblast cells in a T225 flask containing 40 mL of growth media - 36 mL Dulbecco&#39;s Modified Eagle Medium (DMEM), 4 mL of fetal bovine serum (FBS), and 20 &#181;L of penicillin/streptomycin (P/S) - in a CO2 incubator at 37&#176;C. 
	NOTE: Cells should be cultured to approximately 70-80% confluency and should not be allowed to become completely confluent.</li>
	<li>To prepare cells for seeding, rinse the cells with 40 mL of phosphate buffered saline (PBS) twice, then add 5 mL of 0.05% trypsin to detach the cells. Return the cells to the 37 &#176;C CO2 incubator and incubate the cells for 5 min.
	<ol>
		<li>When the cells are fully detached, add 40 mL of growth media to stop the trypsin reaction.</li>
		<li>Count the cells using a hemocytometer or another cell counting device.</li>
	</ol>
	</li>
	<li>Seed 2 mL of cells at 1x105 cells/mL into 35 mm collagen-coated glass-bottom dishes. Incubate the cells overnight in a CO2 incubator at 37 &#176;C. 
	NOTE: Cells should be approximately 50-60% confluent the next day.</li>
	<li>Remove the growth media and replace it with 2 mL of serum-deprived media (growth media without FBS).</li>
	<li>Transfect the cells with full-length dysferlin plasmid using lipotransfection.
	<ol>
		<li>Prepare 150 &#181;L of serum-deprived media in a 1.5 mL tube (one tube for each dish to be transfected).</li>
		<li>Add 3 &#181;L of transfection reagent to each 1.5 mL tube containing serum-deprived media. Incubate it at least 5 min at room temperature.</li>
		<li>In a separate 1.5 mL tube, add 175 &#181;L of serum-deprived media and 3.5 &#181;g of plasmid DNA. 
		NOTE: Multiply the above amounts for each additional dish to be transfected.</li>
		<li>Combine 150 &#181;L of media containing plasmid DNA into a tube with 150 &#181;L of serum-deprived media and transfection reagent. Incubate it for 20 min at room temperature.</li>
		<li>Evenly distribute 300 &#181;L of media with plasmid and transfection reagent across the dish. Incubate the dishes for 24 h in a CO2 incubator at 37&#176;C.</li>
	</ol>
	</li>
	<li>Replace the media with fresh growth media containing serum and incubate it for an additional 24 h in a CO2 incubator at 37 &#176;C.</li>
</ol>
2. Preparation of cells for two-photon wounding assay
<ol>
	<li>Remove the media by pipetting and rinse the cells once with 1 mL of Tyrode&#39;s solution, then add 1 mL of fresh Tyrode&#39;s solution containing 1 &#181;L of 2.5 mM FM 4-64 dye. 
	NOTE: The final concentration of FM 4-64 dye is 2.5 &#181;M.</li>
	<li>If assessment of membrane repair in a calcium-depleted environment is desired, rinse cells with 1 mL of PBS and then add 1 mL of PBS containing 1 mM EGTA. 
	NOTE: As PBS will cause the cells to detach over time, the wounding assay should be completed as quickly as possible.</li>
</ol>
3. Two-photon laser wounding assay
<ol>
	<li>Using an inverted confocal microscope and associated program software, create a 0.2 &#181;m x 2 &#181;m target and place it at the edge of the cell membrane so that the target line overlaps and lies perpendicular to the direction of the cell membrane (Figure 2) .</li>
	<li>Use a 543-nm HeNe laser to excite FM dye signal and set the detection range for 600-760 nm. Use a 488-nm Argon laser to excite GFP signal (Figure 4) and set the detection range for 500-550 nm.
	<ol>
		<li>Create a 5 min time series of sequential image scans, imaging cells every 5 s.</li>
		<li>To generate a membrane lesion, bleach cells using a two-photon laser set to 820-nm, using 15% laser power with 10 iterations and bleach cells 25 s after the beginning of the time series.</li>
	</ol>
	</li>
	<li>To quantify FM 4-64 dye fluorescence, use the software to draw a 6 &#181;m x 6 &#181;m region of interest (ROI) and place it adjacent to the wounding location and within the cell (Figure 3). 
	NOTE: Avoid sampling areas of pixel oversaturation by using the &#34;Range Indicator&#34; function in the software - in this application, red pixels represent oversaturated pixels. Export raw fluorescence values into an electronic spreadsheet for statistical analysis.</li>
	<li>Calculate relative fluorescence values for each time point by subtracting the background value (mean fluorescence value of timepoints prior to membrane wounding) and dividing the net increase by the fluorescence value at t = 0 (Figure 5, 6).</li>
</ol>

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The authors have nothing to disclose.

Two-photon laser wounding of the cell membrane is a precise and versatile technique for assessing the dynamics of membrane resealing in vitro. In this article, we described a protocol for determining cell membrane resealing ability in dysferlinopathy patient cells using the two-photon laser wounding assay. Our results show that dysferlinopathy patient cells are defective in cell membrane resealing, which is consistent with findings by other researchers and which further highlights the striking similarities in cell membrane resealing dynamics between muscle and non-muscle cell types3,11,15,22. We also observed that patient cells transfected with a plasmid containing the full-length dysferlin sequence were rescued from their defective membrane resealing phenotype, demonstrating that full-length dysferlin plasmid can rescue phenotype in non-muscle cell types as well as in muscle27. 
Together, our results reinforce the feasibility and reliability of the two-photon membrane wounding assay as a gold-standard in measuring cell membrane resealing ability in vitro. However, the cost and availability associated with two-photon microscopy could be a potential limiting factor for some research groups seeking to utilize the two-photon membrane wounding assay. Furthermore, our setup utilizes an inverted confocal microscope, which is well-suited to in vitro work but may be less appropriate for in vivo imaging.
It is important to note that the precision of the two-photon laser alignment is critical to the success of the technique. In our experience, troubleshooting of laser alignment was the most common issue encountered. Other issues which could affect the ability to score successful hits during laser wounding include the levelling of the glass-bottom dish on the stage, as well as the orientation of the cells in solution. 
The assay is applicable to current and future studies investigating disease states in which cell membrane integrity or resealing is perturbed, and the efficacy of therapies, such as gene therapy and antisense oligonucleotide-based therapy, which would benefit from having an effective assay for characterizing membrane dynamics so that novel therapeutic options can be explored28,29.

The cell membrane of eukaryotic cells consists of a double-layer of protein-studded phospholipids which defines the intra/extracellular environment of the cell and is essential for maintaining cellular homeostasis and cell survival. Cell membrane injuries arising from mechanical or chemical insults are commonplace in various mammalian cell types, including skeletal and cardiac muscle, stomach, and lung cells1,2,3,4. In addition to injuries consequent to daily physiological function, cell membranes can also be damaged by environmental insults, bacterial toxins, and ischemic reperfusion5. Failure to reseal ruptures in the cell membrane leads to an unregulated influx of extracellular Ca2+- along with other potentially toxic extracellular components - into the cell, triggering downstream signal cascades which can quickly result in cell death1,4,5,6.
To date, there have been several proposed models for membrane repair in cells. Different repair mechanisms may be activated depending on the size and nature of the membrane rupture. For example, it is suggested that cell membranes may utilize lateral flow or protein clogging to repair small disruptions (&#60;1 nm). The lateral fusion model proposes that membrane ruptures are rapidly mended through lateral recruitment of dysferlin-containing membrane7, while the protein clogging model suggests that small perforations are mended through protein aggregations (mostly annexins)8. Conversely, larger membrane lesions trigger Ca2+-dependent, dysferlin-mediated vesicle fusion and formation of a repair patch. In the repair patch model, a rapid Ca2+ influx into the cell triggers the recruitment of multiple proteins (dysferlin, annexins, mitsugumin-53, and EDH proteins) which form a complex or &#34;patch&#34;, along with a fusion of intracellular vesicles or lysosomes at the site of membrane damage4,9,10,11,12. It is important to note that these models are not necessarily mutually exclusive and may work in concert to facilitate membrane repair. Failure to properly reseal cell membrane damage is associated with multiple disease states, including muscular dystrophy (dysferlinopathy - including Miyoshi myopathy, limb-girdle muscular dystrophy type IIB, and distal myopathy with anterior tibial onset)13, cardiomyopathy14, and Chediak-Higashi syndrome (CHS)15.
Given that proper cell membrane integrity and resealing plays such an important role in health and disease, a deeper understanding of the underlying molecular mechanisms of cell membrane repair would be beneficial in the search for novel therapeutic strategies. It is, therefore, necessary to possess appropriate experimental techniques to&#160;monitor the kinetics and evaluate the ability of cell membrane repair. Several in vitro methods for modeling membrane repair have been designed. One strategy involves mechanical injury, which can be facilitated through cell scraping with a pipette/surgical blade/razor or by rolling glass beads over the cells16. However, this type of mechanical injury generates larger lesions, and creates a high degree of variation in cell injury, both within and between cultures.
Another method of generating membrane wounds is laser ablation by two-photon microscopy. In contrast to traditional laser confocal microscopy that employs single photon excitation, the two-photon laser simultaneously uses two long-wavelength, low-energy photons to facilitate the excitation of a high-energy electron17. This non-linear process results in excitation exclusively within the focal plane and not along the entire light path17 (Figure 1). This reduced excitation volume helps to minimize photodamage when imaging live cells18,19. Researchers are therefore able to generate precise lesions in the cell membrane and monitor membrane resealing in real time by using fluorescent dyes and observing changes in the fluorescence intensity as the cell membrane ruptures and then reseals.
This approach has been used repeatedly to study membrane wounding in vitro and in vivo and in multiple cell types20,21. For example, membrane repair defects in fibroblasts and myotubes derived from dysferlinopathy patients were assessed using this technique22,23. Also, single muscle fibers isolated from mice were used to monitor repair patch formation13,24,25. The movement of fluorescently tagged proteins can also be observed during membrane repair in single muscle fibers9. Furthermore, the process of sarcolemmal repair following two-photon laser wounding in zebrafish embryos can be observed in real-time in vivo26.
In this article, we outline a methodology for assessing cell membrane repair dynamics in fibroblasts using two-photon laser wounding, although this methodology can be applied to various cell types for the purpose of quantifying plasma membrane resealing ability in vitro. In this method, cells are incubated with FM4-64, a lipophilic, cell-impermeable dye which quickly fluoresces as it binds to negatively charged phospholipids within the cytoplasm upon entering the cell through the membrane lesion (Figure 2&#38;3). Quantification of dye fluorescence adjacent to the membrane lesion allows for monitoring the time that it takes for the cell membrane to reseal itself. To exemplify the utility of this method, we use dysferlinopathy patient fibroblasts transfected with GFP-conjugated full-length dysferlin (DYSF) plasmids to assess the rescue of cell membrane repair.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher</td><td>11320033</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F1051</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher</td><td>15140122</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Thermo Fisher</td><td>25300062</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>35mm collagen-coated glass-bottom dishes</td><td>MatTek</td><td>P53GCOL-1.5-14-C</td><td></td></tr><tr><td>Serum-deprived media</td><td>Thermo Fisher</td><td>31985070</td><td></td></tr><tr><td>Transfection reagent</td><td>Thermo Fisher</td><td>15338100</td><td></td></tr><tr><td>FM 4-64 Dye</td><td>Invitrogen</td><td>T13320</td><td></td></tr><tr><td>Tyrode&amp;rsquo;s Salts Solution</td><td>Sigma-Aldrich</td><td>T2397</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Carl Zeiss</td><td>NA</td><td></td></tr><tr><td>Chameleon Two-photon laser</td><td>Coherent</td><td>NA</td><td></td></tr></tbody></table>

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.

Healthy human fibroblasts, non-treated dysferlinopathy patient fibroblasts, and patient fibroblasts transfected with a plasmid containing the full-length dysferlin sequence were subjected to two-photon laser wounding to assess membrane resealing ability in real time. Healthy human fibroblast cells displayed low levels of FM 4-64 fluorescence activation following laser wounding and non-treated patient fibroblasts exhibited a high degree of relative fluorescence intensity following injury (Figure 5&#38;6). Patient cells that were transfected with full-length dysferlin plasmid showed reduced relative FM 4-64 fluorescence intensity compared with non-treated patient controls and were comparable with fluorescence values observed in the healthy control (Figure 5&#38;6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56999/56999fig1.jpg" /> 
Figure 1. One-photon versus two-photon microscopy. In a traditional one-photon microscope system, a single high energy photon (UV or visible spectrum) is used to excite a fluorophore (A). In a two-photon system, two lower-energy photons (near infrared) are used to excite a fluorophore (B). Depicted within the cylinders is the fluorescence signal generated throughout the focal plane, demonstrating how the two-photon laser system can focus excitation light to within a specific region (B) and exclude light from out-of-focus planes (A). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56999/56999fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56999/56999fig2.jpg" /> 
Figure 2. Placing a target at the cell membrane. Using software, a 0.2 &#181;m x 2 &#181;m target is drawn and placed overlapping and perpendicular to the cell membrane.The arrow indicates the target. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56999/56999fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56999/56999fig3.jpg" /> 
Figure 3. Fluorescence signal from FM dye as it infiltrates the cell through membrane rupture. As the cell membrane ruptures, lipophilic FM 4-64 dye infiltrates the cell and fluoresces as it binds negatively-charged phospholipids within the cytoplasm. Using software, a region of interest (ROI) can be drawn (white box) and fluorescence values within the box can be used to calculate relative fluorescence changes within the cell. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56999/56999fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56999/56999fig4.jpg" /> 
Figure 4. Fluorescence signal from GFP-conjugated full-length DYSF plasmid. Following plasmid transfection, patient fibroblast cells express full-length DYSF protein. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56999/56999fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56999/56999fig5.jpg" /> 
Figure 5. Quantification of fluorescence over time. Fluorescence values calculated by computer software from within the ROI can be used to determine relative fluorescence changes over time for each treatment group. The arrow indicates the time of laser ablation. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56999/56999fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56999/56999fig6.jpg" /> 
Figure 6. Change in relative fluorescence over time. Relative fluorescence (RF) values for each time point are calculated by subtracting the background fluorescence value (calculated as the mean of the fluorescence values from the timepoints prior to membrane wounding) from the given fluorescence values (Signal Intensity - Background = &#916;F) and dividing the net increase (&#916;F) by the fluorescence value at t = 0 (F0). Therefore, RF = &#916;F/F0. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56999/56999fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Cell Membrane Repair Assay Using a Two-photon Laser Microscope at JoVE.com

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

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 ...

cell-membrane-repair-assay-using-a-two-photon-laser-microscope

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

12.3K Views.  University of Alberta Faculty of Medicine and Dentistry. 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.

Video: Cell Membrane Repair Assay Using a Two-photon Laser Microscope

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

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Genetics

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.

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.

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 ...

Biology

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 ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

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 ...

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. ...

Two-Photon Laser-Induced Neural Injury: A Method to Observe Axon Degeneration and Regeneration in Drosophila Larvae

Source: Li, D., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/57557/" target="_blank">A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System</a>. J. Vis. Exp. (2018).
This video describes two-photon laser ablation of axons in a Drosophila melanogaster larva and an example protocol to induce injury and image the injury site.

Source: Li, D., et al. A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System. J. Vis. Exp. 
...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Larva

An In Vitro Fluorescence-Based Assay to Measure Plasma Membrane Resealing Efficiency

To set up a high throughput fluorescence-based assay for the repair permissive conditions, wash the cells two times with 200 microliters of 37 degrees Celsius M1 medium per well, and add 100 microliters of fresh 37 degrees Celsius M1 medium supplemented with 30-micromolar propidium iodide after discarding the second wash.
For repair restrictive conditions, wash the cells one time with 200 microliters of 37 degrees Celsius M2 medium supplemented with 5-millimolar EGTA per well, followed by one wash with 200 microliters of M2 medium alone.
After discarding the second wash, add 100 microliters of fresh 37 degrees Celsius M2 medium supplemented with 30-micromolar propidium iodide per well. Then, image the plate under transmitted light, GFP, and PI, as just demonstrated. While the plate is being imaged, place a 96-well round-bottom polypropylene microplate on ice, and configure the plate as just demonstrated for the previous plate.
For repair permissive conditions, add 100 microliters of ice-cold M1 medium supplemented with 60 micromolar propidium iodide per well, followed by the addition of 100 microliters of ice-cold M1 with or without 4x listeriolysin O per well.
For repair restrictive conditions, add 100 microliters of ice-cold M2 medium supplemented with 60 micromolar propidium iodide per well, followed by the addition of 100 microliters of ice-cold M2 with or without 4x listeriolysin O per well.
It is imperative to prepare listeriolysin O at the appropriate experimental concentration on ice approximately 5 minutes prior to the start of the kinetic assay when plate 1 is on ice and plate 2 is being prepared.
When the first plate is finished imaging, immediately transfer the plate onto a sheet of aluminum foil on ice. After 5 minutes, transfer 100 microliters from each well of the second plate to the appropriate corresponding well in the first cooled plate, inserting the pipette tips below the meniscus of solution in each well before ejecting the volume without mixing for proper distribution of the toxin.
Allow the toxin to bind to the host cells for 1 minute, and immediately transfer the first plate to the plate reader for the postkinetic assay using the spectrofluorometer mode. At the end of the kinetic assay, immediately acquire a postkinetic imaging of the plate image as demonstrated for the prekinetic imaging.

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Summary

Explore More Videos

Chapters in this video

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

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

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

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

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

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

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

Two-Photon Laser-Induced Neural Injury: A Method to Observe Axon Degeneration and Regeneration in Drosophila Larvae

An In Vitro Fluorescence-Based Assay to Measure Plasma Membrane Resealing Efficiency