Edited by: Silvia Mercedes Uriarte, University of Louisville, United States
Reviewed by: Leigh A. Knodler, Washington State University, United States; Esther Orozco, Centro de Investigación y de Estudios Avanzados del IPN, Mexico
*Correspondence: Stephanie M. Seveau
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The plasma membrane of mammalian cells is susceptible to disruption by mechanical and biochemical damages that frequently occur within tissues. Therefore, efficient and rapid repair of the plasma membrane is essential for maintaining cellular homeostasis and survival. Excessive damage of the plasma membrane and defects in its repair are associated with pathological conditions such as infections, muscular dystrophy, heart failure, diabetes, and lung and neurodegenerative diseases. The molecular events that remodel the plasma membrane during its repair remain poorly understood. In the present work, we report the development of a quantitative high-throughput assay that monitors the efficiency of the plasma membrane repair in real time using a sensitive microplate reader. In this assay, the plasma membrane of living cells is perforated by the bacterial pore-forming toxin listeriolysin O and the integrity and recovery of the membrane are monitored at 37°C by measuring the fluorescence intensity of the membrane impermeant dye propidium iodide. We demonstrate that listeriolysin O causes dose-dependent plasma membrane wounding and activation of the cell repair machinery. This assay was successfully applied to cell types from different origins including epithelial and muscle cells. In conclusion, this high-throughput assay provides a novel opportunity for the discovery of membrane repair effectors and the development of new therapeutic compounds that could target membrane repair in various pathological processes, from degenerative to infectious diseases.
The repair of the plasma membrane is a fundamental process that maintains cell homeostasis, prevents the loss of difficult to replace cells (e.g., cardiac myocytes or neurons) and eliminates the need for replacing frequently injured cells. Mechanical stress and molecules that can directly damage the plasma membrane are major causes of cell injuries. Membrane injuries due to mechanical wounding frequently occur in contractile tissues (McNeil and Khakee,
The objective of this work was to develop a high-throughput, microplate-based assay to assess the repair efficiency of the plasma membrane of mammalian cells. Different models for the plasma membrane repair process have been proposed; they all consider that the influx of extracellular Ca2+, through the site of injury, is a trigger for activation of the repair process. The proposed repair processes include the decrease in membrane surface tension via dissociation of the cortical F-actin network, patching of the membrane lesions by fusion of intracellular vesicles with the plasma membrane, regrowth and contraction of F-actin at the edges of the lesion to close the wound, removal of membrane lesions by endocytosis, shedding of the lesion-containing membrane, and exocytosis of enzymes that degrade the agents responsible for membrane attack (e.g., pore-forming toxins) (Bi et al.,
Several approaches are used to model the mechanical cell wounding that normally occurs under physiologically strenuous conditions in skeletal muscles, heart, lungs, or intestines. These approaches consist of inducing contraction or stretching of a cell monolayer grown on a flexible surface, cell scraping from the dish, or creating membrane abrasions with glass beads (Liu et al.,
Plasma membrane wounding can also be achieved by adding pore-forming agents such as bacterial toxins to the cell culture medium. The size of the membrane pores can vary from 1 to 50 nm depending upon the toxin. Small pores such as those formed by aerolysin (produced by
To establish the efficiency of plasma membrane repair, most approaches rely on the quantification of plasma membrane integrity using membrane impermeant dyes. Those include Trypan blue, propidium iodide, and FM-dyes, which can penetrate wounded cells leading to a change in cell color or fluorescence (Cochilla et al.,
Quantitative fluorescence microscopy and flow-cytometry can be used to measure the uptake of fluorescent dyes by damaged cells. The advantage of flow cytometry is the rapid measurement of large cell populations (Idone et al.,
For wounding the plasma membrane, cells were exposed to recombinant six His-tagged-listeriolysin O (LLO), purified as previously described (Vadia et al.,
We selected two mammalian cell lines, HeLa (ATCC #CCL-2) and C2C12 (ATCC #CRL-1772), which have been frequently used in studies assessing the mechanisms of membrane repair. HeLa cells are of human epithelial origin and were used to study membrane repair following damage by either mechanical or biological injuries (Idone et al.,
The assays involved cells cultured in 96-well plates and kinetics were performed at 37°C for 30 min with fluorescence measurements at 5 min time intervals. Both the duration and time intervals can be modified based on experimental needs. The minimum time interval for measurement of a full 96-well-plate with the Spectra Max i3x Multi-Mode Detection Platform (Molecular devices) is 30 s. Two different plate types were used in these studies. Plate 1: Corning® 96-well flat clear bottom black polystyrene TC-treated microplates, individually wrapped, with lid and sterile (#3603). Plate 2: Nunc™ 96-well polystyrene round bottom sterile plates (#262162).
Cells were plated in a 96-well plate (plate 1) in triplicate for each experimental condition, in 200 μl of their respective culture medium (HeLa: 2.5 × 104 cells/well or C2C12: 1 × 104 cells/well). The following day, cells in plate 1 were pre-incubated with the indicated concentrations of cytochalasin D (Sigma) or equivalent amounts of the DMSO vehicle for 10 min at 37°C. Cytochalasin D, or control DMSO, was maintained at the same concentration in the assay buffers (
The baseline normalized individual data was first log 10 transformed to reduce skewness and variance, and then the mean of the triplicates of each independent experiment was used for analysis with linear mixed effects models to take account of the correlation among the observations from the same independent replicate. In order to test whether the speed of fluorescence intensity change over time is significantly different between the lowest concentration (0.1 nm) and other higher concentrations within the same treatment condition, as well as the same concentration between the two conditions (M1 and M2), trend of fluorescence intensity change over time was compared from the linear mixed models. In addition, the mean fluorescence intensity averaged across the measurement time was also compared between the aforementioned groups to investigate whether the higher concentrations induced overall higher fluorescence intensity than the 0.1 nM concentration and whether there is difference between M1 and M2 conditions for the same concentration used. The fluorescence intensity was also compared among the aforementioned groups at time = 30 min using an ANOVA model. Holm's procedure was used to adjust for multiple testing and the adjusted
To measure the efficiencies of plasma membrane wounding and repair following exposure to LLO (0.1–2 nM), we incubated HeLa cells with the fluorescent dye propidium iodide (PI) in media supplemented (M1), or not (M2), with CaCl2. In the absence of extracellular Ca2+ (M2), cells cannot repair their plasma membrane and consequently, the PI fluorescence intensities reflected the extent of cell wounding by LLO. In the presence of extracellular Ca2+ (M1), cells can undergo repair and the PI fluorescence intensities reflected the contributions of both cell wounding by LLO and repair. Importantly, formation of the LLO pore is a Ca2+-independent process (Arnett et al.,
Representative experiment of membrane wounding by LLO and repair kinetics in HeLa cells. HeLa cells were exposed to the indicated concentrations of LLO in M1 (+ 1.2 mM CaCl2, solid lines) or M2 (without CaCl2, dashed lines) containing 30 μM PI and incubated in the plate reader at 37°C for 30 min. Fluorescence intensities were measured every 5 min. The baseline fluorescence levels of control cells incubated without LLO, at each time point in M1 and in M2, were subtracted from the values obtained with cells incubated with LLO. Data are the average fluorescence intensities expressed in arbitrary unit ± standard deviations (S.D.) of triplicates for each experimental condition.
LLO induces dose-dependent membrane wounding and repair in HeLa cells. HeLa cells were exposed to the indicated concentrations of LLO in M1 (solid lines)
Fluorescence intensity change over time (trend) was compared between LLO concentrations higher than 0.1 nM and that of 0.1 nM for M1 and M2 from a linear mixed effects model.
Dose Effect | 2.7087 | 0.0002 | 1.2845 | 4.1328 |
M1 comparing LLO 2 to 0.1 | 0.1487 | <0.0001 | 0.09871 | 0.1986 |
M1 comparing LLO 1 to 0.1 | 0.1448 | <0.0001 | 0.09480 | 0.1947 |
M1 comparing LLO 0.5 to 0.1 | 0.1557 | <0.0001 | 0.1058 | 0.2057 |
M1 comparing LLO 0.25 to 0.1 | 0.03857 | 0.1297 | −0.01140 | 0.08853 |
M2 comparing LLO 2 to 0.1 | 0.006204 | 0.8070 | −0.04376 | 0.05617 |
M2 comparing LLO 1 to 0.1 | 0.01519 | 0.5498 | −0.03477 | 0.06516 |
M2 comparing LLO 0.5 to 0.1 | 0.02558 | 0.3143 | −0.02438 | 0.07554 |
M2 comparing LLO 0.25 to 0.1 | 0.01501 | 0.5546 | −0.03495 | 0.06497 |
Mean fluorescence intensities averaged across time were compared between LLO concentrations higher than 0.1 and 0.1 nM within M1 and M2, as well as between M1 and M2 for the same dose, where the estimate is the difference between condition 1 LLO dose 1 and condition 2 LLO dose 2.
M1 | 0.1 | M1 | 0.25 | −0.6610 | 0.0097 | −1.1606 | −0.1614 |
M1 | 0.1 | M1 | 0.5 | −2.4562 | <0.0001 | −2.9559 | −1.9566 |
M1 | 0.1 | M1 | 1 | 3.0816 | <0.0001 | 2.5820 | 3.5813 |
M1 | 0.1 | M1 | 2 | 3.2959 | <0.0001 | 2.7963 | 3.7955 |
M2 | 0.1 | M2 | 0.25 | −0.6223 | 0.0148 | −1.1220 | −0.1227 |
M2 | 0.1 | M2 | 0.5 | −0.7325 | 0.0042 | −1.2322 | −0.2329 |
M2 | 0.1 | M2 | 1 | 1.0507 | <0.0001 | 0.5511 | 1.5503 |
M2 | 0.1 | M2 | 2 | 1.1092 | <0.0001 | 0.6096 | 1.6089 |
M1 | 0.1 | M2 | 0.1 | −2.6650 | <0.0001 | −3.1646 | −2.1654 |
M1 | 0.25 | M2 | 0.25 | −2.6263 | <0.0001 | −3.1260 | −2.1267 |
M1 | 0.5 | M2 | 0.5 | −0.9413 | 0.0003 | −1.4409 | −0.4417 |
M1 | 1 | M2 | 1 | −0.6341 | 0.0131 | −1.1337 | −0.1344 |
M1 | 2 | M2 | 2 | −0.4784 | 0.0605 | −0.9780 | 0.02126 |
Representative images of HeLa cells. Phase contrast and fluorescence images of HeLa cells were acquired with a 4X objective located within the plate reader at the end of the kinetic assay corresponding to time the point 30 min.
To validate the use of this assay for the identification of molecules that affect the repair machinery, we treated cells with the drug cytochalasin D (CD). This drug induces the disassembly of F-actin by binding to the barbed end of actin filaments. Cell treatment with low concentrations of CD has been shown to slightly enhance the membrane repair efficiency of cells exposed to LLO (Vadia et al.,
Effect of F-actin disassembly on plasma membrane repair. HeLa cells were pre-incubated in the presence of 5 μM cytochalasin D (CD, stock solution was stored in DMSO) for 10 min at 37°C, and the drug was maintained at the same concentration throughout the assay. Cells were exposed to 0.5 nM LLO, or not, in M1 or M2 supplemented with 30 μM PI. Control cells were incubated with a dilution of vehicle (DMSO) similar to cells treated with CD. Data are the average of four independent experiments, each performed in triplicate, and the error bars represent the standard error of mean (SEM). Statistical analyses correspond to the trend comparison (T,
Fluorescence intensity change over time (trend) was compared between the indicated experimental conditions from a linear mixed effects model.
Comparing LLO (M2) to LLO (M1) | −0.00710 | 0.02206 | 0.7478 | −0.0507 | 0.03647 |
Comparing LLO (M1) and LLO (M1) + CD | 0.1088 | 0.02206 | <0.0001 | 0.06521 | 0.1524 |
Comparing LLO (M1) + CD and (M1) + CD | 0.09729 | 0.02206 | <0.0001 | 0.05372 | 0.1409 |
Mean fluorescence intensities averaged across time were compared between the experimental conditions 1 and 2, where the estimate is the difference between condition 1 and condition 2.
LLO (M1) | LLO (M2) | −0.7887 | 0.0005 | −1.2244 | −0.3530 |
LLO (M1) | LLO (M1) + CD | 1.1448 | <0.0001 | 0.7091 | 1.5805 |
LLO (M1) + CD | (M1) + CD | 1.4408 | <0.0001 | 1.0051 | 1.8765 |
We evaluated the applicability of the assay to membrane repair in muscle cells. We exposed C2C12 cells to various concentrations of LLO, as performed previously with Hela cells. Data presented in Figure
Membrane wounding and repair in muscle cells. C2C12 cells were exposed to the indicated concentrations of LLO in M1
Fluorescence intensity change over time (trend) was compared between LLO concentrations higher than 0.1 nM and that of 0.1 nM for M1 and M2 from a linear mixed effects model.
Dose effect | 2.4171 | <0.0001 | 1.3080 | 3.5262 |
M1 comparing LLO 2 to 0.1 | 0.1072 | <0.0001 | 0.06825 | 0.1461 |
M1 comparing LLO 1 to 0.1 | 0.09803 | <0.0001 | 0.05912 | 0.1369 |
M1 comparing LLO 0.5 to 0.1 | 0.09391 | <0.0001 | 0.05501 | 0.1328 |
M1 comparing LLO 0.25 to 0.1 | 0.04633 | 0.0198 | 0.007420 | 0.08524 |
M2 comparing LLO 2 to 0.1 | −0.01475 | 0.4559 | −0.05366 | 0.02416 |
M2 comparing LLO 1 to 0.1 | −0.01224 | 0.5360 | −0.05115 | 0.02667 |
M2 comparing LLO 0.5 to 0.1 | −0.00343 | 0.8624 | −0.04234 | 0.03548 |
M2 comparing LLO 0.25 to 0.1 | 0.001384 | 0.9442 | −0.03753 | 0.04029 |
Mean fluorescence intensities averaged across time were compared between LLO concentrations higher than 0.1 and 0.1 nM within M1 and M2, as well as between M1 and M2 for the same dose, where the estimate is the difference between condition 1 LLO dose 1 and condition 2 LLO dose 2.
M1 | 0.1 | M1 | 0.25 | −1.2962 | <0.0001 | −1.6853 | −0.9071 |
M1 | 0.1 | M1 | 0.5 | −1.4885 | <0.0001 | −1.8776 | −1.0994 |
M1 | 0.1 | M1 | 1 | −2.0664 | <0.0001 | −2.4555 | −1.6773 |
M1 | 0.1 | M1 | 2 | −2.3735 | <0.0001 | −2.7626 | −1.9845 |
M2 | 0.1 | M2 | 0.25 | −0.7891 | <0.0001 | −1.1782 | −0.4000 |
M2 | 0.1 | M2 | 0.5 | −1.0147 | <0.0001 | −1.4038 | −0.6256 |
M2 | 0.1 | M2 | 1 | −1.1049 | <0.0001 | −1.4940 | −0.7158 |
M2 | 0.1 | M2 | 2 | −1.1723 | <0.0001 | −1.5614 | −0.7832 |
M1 | 0.1 | M2 | 0.1 | −1.6531 | <0.0001 | −2.0422 | −1.2640 |
M1 | 0.25 | M2 | 0.25 | −1.1460 | <0.0001 | −1.5351 | −0.7569 |
M1 | 0.5 | M2 | 0.5 | −1.1793 | <0.0001 | −1.5684 | −0.7902 |
M1 | 1 | M2 | 1 | 0.6915 | 0.0005 | −1.0806 | −0.3024 |
M1 | 2 | M2 | 2 | −0.4518 | 0.1230 | −0.8409 | −0.06269 |
Mean fluorescence intensities averaged across time were compared between various LLO concentrations between M1 and M2, where the estimate is the difference between condition 1 LLO dose 1 and condition 2 LLO dose 2.
0.1 | M1 | 0.25 | M1 | −2.2701 | 0.0001 | −3.3177 | −1.2225 |
0.1 | M1 | 0.5 | M1 | −2.6468 | <0.0001 | −3.6944 | −1.5991 |
0.1 | M1 | 1 | M1 | −3.1918 | <0.0001 | −4.2394 | −2.1441 |
0.1 | M1 | 2 | M1 | −3.4781 | <0.0001 | −4.5257 | −2.4305 |
0.1 | M2 | 0.25 | M2 | −0.7081 | 0.1769 | −1.7557 | 0.3396 |
0.1 | M2 | 0.5 | M2 | −0.8450 | 0.1095 | −1.8926 | 0.2027 |
0.1 | M2 | 1 | M2 | −0.8510 | 0.1071 | −1.8986 | 0.1966 |
0.1 | M2 | 2 | M2 | −0.8843 | 0.0947 | −1.9320 | 0.1633 |
0.1 | M1 | 0.1 | M2 | −2.6866 | <0.0001 | −3.7342 | −1.6389 |
0.25 | M1 | 0.25 | M2 | −1.1245 | 0.0364 | −2.1722 | −0.07690 |
0.5 | M1 | 0.5 | M2 | −0.8848 | 0.0945 | −1.9324 | 0.1629 |
1 | M1 | 1 | M2 | −0.3458 | 0.5040 | −1.3934 | 0.7018 |
2 | M1 | 2 | M2 | −0.09279 | 0.8572 | −1.1404 | 0.9548 |
We report in this manuscript a sensitive and high-throughput assay to study plasma membrane repair. This assay can be used with various adherent cell types such as epithelial cells (Figures
Some plate readers are equipped with a microscope objective allowing phase-contrast and fluorescence imaging of cells as their fluorescence intensity is measured. This allows for the determination of cell density and if distinct cell populations co-exist in a given experimental condition. In particular, we noticed that in experimental conditions preventing plasma membrane repair, nearly all cells showed uptake of the PI dye. Conversely, under conditions that allowed for membrane repair, we could observe a population of PI negative cells that had presumably efficiently resealed their plasma membrane (Figure
It should be noted that while our assay measured fluorescence intensities at 5 min time intervals for a total of 30 min, the standard read time for a 96-well plate is 25–30 s; therefore, the assay can be adapted to perform kinetics with shorter time intervals if necessary. Alternatively, this assay could be used as an end point assay (Table
Importantly, the cell density was unaltered in any experimental conditions used in this work. Using distinct cell lines and/or experimental conditions will require ensuring that cells do not detach, which would significantly complicate data interpretation. Thus, we recommend the use of a plate reader that has imaging capability to ensure that cells do not detach. If cell detachment cannot be avoided, a second fluorochrome should be added to estimate cell density. This would allow for correction of the PI intensity based upon the cell density. To damage the plasma membrane, we used the CDC listeriolysin O because our laboratory studies the effects of LLO on mammalian cells. However, other CDCs could be used to replace LLO. Indeed, CDC members are highly homologous and form large transmembrane pores of similar size in cholesterol-rich membranes (Tweten et al.,
Our current knowledge about the membrane repair machinery is still limited. In particular, it is unclear if different damage conditions elicit various repair mechanisms to reseal the plasma membrane (Andrews et al.,
Overall, this assay is a promising tool for the discovery of effectors of the cell repair machinery and for the screening of compounds intended to develop new therapeutic approaches for the treatment of diseases associated with compromised membrane repair capacity.
SPS designed and performed all of the experimental work, prepared the manuscript and figures. XZ performed the statistical analyses and prepared the manuscript and Tables. JGTL edited the manuscript and prepared the figures. NW provided the C2C12 and H9C9 cells and corresponding cell culture methods as well as prepared the manuscript. SMS designed the assay, prepared the manuscript and figures.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by the NIH/NIAID (RO1AI107250) to SMS. This work was sponsored by NIH/NIAID award # 1-T32-AI-112542, a NRSA training grant administered by the Center for Microbial Interface Biology (CMIB) at The Ohio State University (SPS postdoctoral fellowship). We would like to thank Sayak Bhattacharya for helping in culturing the cardiac cell lines, Christopher Phelps, and Lauren Johnson for careful editing of the manuscript.
The Supplementary Material for this article can be found online at:
Representation of the standard deviations of the Figures