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Materials

References

Immunology and Infection

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

Published: January 7th, 2019

DOI:

10.3791/58351

1Department of Microbial Infection and Immunity, The Ohio State University, 2Department of Microbiology, The Ohio State University, 3Infectious Diseases Institute, The Ohio State University, 4Division of Biostatistics, College of Public Health, The Ohio State University

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 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.

Mammalian cells are subject to mechanical, osmotic, and biochemical stress, resulting in the loss of plasma membrane integrity. Without rapid and efficient resealing, damaged cells would quickly succumb to programmed or necrotic death. Since the 1960s, efforts to understand the plasma membrane resealing process have been motivated by the devastating consequences associated with its dysfunctions. Indeed, diseases such as Limb-Girdle Muscular Dystrophy, diabetes, and Chediak-Higashi Syndrome have been linked to deficient plasma membrane repair due to mutations in the gene encoding dysferlin, production of advanced glycation end products, and defects in the lysosomal tra....

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1. Preparation

  1. Cell Plating
    Note: Human cervical epithelial cells, HeLa and HeLa expressing Histone 2B-GFP (H2B-GFP), were used in this protocol, but this assay can be adapted to other mammalian cells19.
    1. Detach adherent cells from a 75 cm2 cell culture flask by washing the cells with 2 mL of Trypsin-EDTA 0.25%. Replace the used trypsin with 2 mL of fresh trypsin-EDTA 0.25%.
    2. Incubate the cells at 37 ˚C for 5 min until the cells have rounded .......

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Cell counting accuracy: HeLa cells are frequently used as a model mammalian cell line to explore membrane repair mechanisms. When assessing membrane repair at the cell population level, it is important to plate cells at the same concentration in all wells for proper data interpretation. It is also important to verify at the time of the assay that cell numbers are equivalent across wells. HeLa cells that constitutively express histone 2B fused to GFP (H2B-GFP) were introduced in this assay.......

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This assay measures the efficiency of membrane resealing at the cell population level with high-throughput capacity. It can be used to screen for cellular components or drug libraries that could affect membrane repair. The described assay used a 96-well plate format, but it can be adapted to 384-well plates for higher throughput. An advantage of this assay is its ability to obtain fluorescence measurements of adherent living cells in real time without the need for excessive cell processing such as cell detachment, fixati.......

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We acknowledge Dr. Jesse Kwiek (The Ohio State University) for kindly allowing us to use his multi-mode detection platform for some preliminary experiments. Research reported in this article was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number RO1AI107250 to Stephanie Seveau. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Name Company Catalog Number Comments
SpectraMax i3x Multi-Mode Microplate Reader Molecular Devices i3x
MiniMax 300 Imaging cytometer Molecular Devices 5024062
TO-PRO-3 ThermoFisher Scientific T3605
Propidium Iodide ThermoFisher Scientific P3566
HeLa ATCC CCL2
HeLa H2B-GFP Millipore SCC117
Trypsin-EDTA 0.25% ThermoFisher Scientific 25200056
96-well Corning flat bottom black polystyrene tissue culture treated plate Corning 3603
Hanks' balanced Salts Sigma-Aldrich H4891
EGTA ISC BioExpress 0732-100G
HEPES Fisher Scientific BP310-500
D-(+)-Glucose, HybriMax Sigma-Aldrich G5146-1KG

  1. Demonbreun, A. R., McNally, E. M. Plasma Membrane Repair in Health and Disease. Current Topics in Membranes. 77, 67-96 (2016).
  2. Howard, A. C., McNeil, A. K., McNeil, P. L. Promotion of plasma membrane repair by vitamin E. Nature Communications. 2, 597 (2011).
  3. Howard, A. C., et al. A novel cellular defect in diabetes: membrane repair failure. Diabetes. 60 (11), 3034-3043 (2011).
  4. Lozano, M. L., et al. Towards the targeted management of Chediak-Higashi syndrome. Orphanet Journal of Rare Diseases. 9, 132 (2014).
  5. Vainzof, M., et al. Dysferlin protein analysis in limb-girdle muscular dystrophies. Journal of Molecular Neuroscience. 17 (1), 71-80 (2001).
  6. Huynh, C., et al. Defective lysosomal exocytosis and plasma membrane repair in Chediak-Higashi/beige cells. Proceeding of the National Academy of Sciences of the United States of America. 101 (48), 16795-16800 (2004).
  7. Cooper, S. T., McNeil, P. L. Membrane Repair: Mechanisms and Pathophysiology. Physiological Reviews. 95 (4), 1205-1240 (2015).
  8. Steinhardt, R. A., Bi, G., Alderton, J. M. J. M. Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. Science. 263 (5145), 390-393 (1994).
  9. De Mello, W. C. Membrane sealing in frog skeletal-muscle fibers. Proceedings of the National Academy of Sciences of the United States of America. 70 (4), 982-984 (1973).
  10. Fishman, H. M., Tewari, K. P., Stein, P. G. Injury-induced vesiculation and membrane redistribution in squid giant axon. Biochimica et Biophysica Acta. 1023 (3), 421-435 (1990).
  11. Davenport, N. R., Bement, W. M. Cell repair: Revisiting the patch hypothesis. Communicative & Integrative Biology. 9 (6), 1253643 (2016).
  12. McNeil, P. L., et al. Patching plasma membrane disruptions with cytoplasmic membrane. Journal of Cell Science. 113 (11), 1891-1902 (2000).
  13. Terasaki, M., Miyake, K., McNeil, P. L. Large plasma membrane disruptions are rapidly resealed by Ca2+-dependent vesicle-vesicle fusion events. Journal of Cell Biology. 139 (1), 63-74 (1997).
  14. Bi, G. Q., Alderton, J. M., Steinhardt, R. A. Calcium-regulated exocytosis is required for cell membrane resealing. Journal of Cell Biology. 131 (6 Pt. 2), 1747-1758 (1995).
  15. Tam, C., et al. Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair. Journal of Cell Biology. 189 (6), 1027-1038 (2010).
  16. Rodriguez, A., et al. Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells. Journal of Cell Biology. 137 (1), 93-104 (1997).
  17. Reddy, A., Caler, E. V., Andrews, N. W. Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes. Cell. 106 (2), 157-169 (2001).
  18. Jimenez, A. J., et al. ESCRT machinery is required for plasma membrane repair. Science. 343 (6174), 1247136 (2014).
  19. Pathak-Sharma, S., et al. High-Throughput Microplate-Based Assay to Monitor Plasma Membrane Wounding and Repair. Frontiers in Cellular and Infection Microbiology. 7, 305 (2017).
  20. Hamon, M. A., et al. Listeriolysin O: the Swiss army knife of Listeria. Trends in Microbiology. 20 (8), 360-368 (2012).
  21. Seveau, S. Multifaceted activity of listeriolysin O, the cholesterol-dependent cytolysin of Listeria monocytogenes. Subcellular Biochemistry. 80, 161-195 (2014).
  22. Osborne, S. E., Brumell, J. H. Listeriolysin O: from bazooka to Swiss army knife. Philosophical Transactions of the Royal Society of London B: Biological Sciences. 372 (1726), (2017).
  23. Lukoyanova, N., Hoogenboom, B. W., Saibil, H. R. The membrane attack complex, perforin and cholesterol-dependent cytolysin superfamily of pore-forming proteins. Journal of Cell Science. 129 (11), 2125-2133 (2016).
  24. Tweten, R. K. Cholesterol-dependent cytolysins, a family of versatile pore-forming toxins. Infection and Immunity. 73 (10), 6199-6209 (2005).
  25. Koster, S., et al. Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation. Nature Communications. 5, 3690 (2014).
  26. Duncan, J. L., Schlegel, R. Effect of streptolysin O on erythrocyte membranes, liposomes, and lipid dispersions. A protein-cholesterol interaction. Journal of Cell Biology. 67 (1), 160-174 (1975).
  27. Morgan, P. J., et al. Subunit organisation and symmetry of pore-forming, oligomeric pneumolysin. FEBS Letters. 371 (1), 77-80 (1995).
  28. Leung, C., et al. Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin. eLife. 3, (2014).
  29. Marchioretto, M., et al. What planar lipid membranes tell us about the pore-forming activity of cholesterol-dependent cytolysins. Biophysical Chemistry. 182, 64-70 (2013).
  30. Palmer, M., et al. Assembly mechanism of the oligomeric streptolysin O pore: the early membrane lesion is lined by a free edge of the lipid membrane and is extended gradually during oligomerization. European Molecular Biology Organization Journal. 17 (6), 1598-1605 (1998).
  31. Bavdek, A., et al. pH dependence of listeriolysin O aggregation and pore-forming ability. Federation of European Biochemical Society Journal. 279 (1), 126-141 (2012).
  32. Schuerch, D. W., Wilson-Kubalek, E. M., Tweten, R. K. Molecular basis of listeriolysin O pH dependence. Proceeding of the National Academy of Sciences of the United States of America. 102 (35), 12537-12542 (2005).
  33. Cassidy, S. K., O'Riordan, M. X. More than a pore: the cellular response to cholesterol-dependent cytolysins. Toxins (Basel). 5 (4), 618-636 (2013).
  34. Lam, J., et al. Host cell perforation by listeriolysin O (LLO) activates a Ca(2+)-dependent cPKC/Rac1/Arp2/3 signaling pathway that promotes L. monocytogenes internalization independently of membrane resealing. Molecular Biology of the Cell. , (2017).
  35. Gekara, N. O., Weiss, S. Lipid rafts clustering and signalling by listeriolysin O. Biochemical Society Transactions. 32 (Pt 5), 712-714 (2004).
  36. Magassa, N., Chandrasekaran, S., Caparon, M. G. Streptococcus pyogenes cytolysin-mediated translocation does not require pore formation by streptolysin O. European Molecular Biology Organization Reports. 11 (5), 400-405 (2010).
  37. Baba, H., et al. Induction of gamma interferon and nitric oxide by truncated pneumolysin that lacks pore-forming activity. Infection and Immunity. 70 (1), 107-113 (2002).
  38. Carrero, J. A., Vivanco-Cid, H., Unanue, E. R. Listeriolysin o is strongly immunogenic independently of its cytotoxic activity. Public Library of Science One. 7 (3), e32310 (2012).
  39. Coconnier, M. H., et al. Listeriolysin O-induced stimulation of mucin exocytosis in polarized intestinal mucin-secreting cells: evidence for toxin recognition of membrane-associated lipids and subsequent toxin internalization through caveolae. Cell Microbiology. 2 (6), 487-504 (2000).
  40. Suzuki, T., et al. DNA staining for fluorescence and laser confocal microscopy. Journal of Histochemistry and Cytochemistry. 45 (1), 49-53 (1997).
  41. Bink, K., et al. TO-PRO-3 is an optimal fluorescent dye for nuclear counterstaining in dual-colour FISH on paraffin sections. Histochemistry and Cell Biology. 115 (4), 293-299 (2001).
  42. Zhang, J. H., Chung, T. D., Oldenburg, K. R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. Journal of Biomolecular Screening. 4 (2), 67-73 (1999).
  43. Birmingham, A., et al. Statistical methods for analysis of high-throughput RNA interference screens. Nature Methods. 6 (8), 569-575 (2009).
  44. Zhang, X. D. A pair of new statistical parameters for quality control in RNA interference high-throughput screening assays. Genomics. 89 (4), 552-561 (2007).
  45. Zhang, X. D. A new method with flexible and balanced control of false negatives and false positives for hit selection in RNA interference high-throughput screening assays. Journal of Biomolecular Screening. 12 (5), 645-655 (2007).
  46. Idone, V., et al. Repair of injured plasma membrane by rapid Ca2+-dependent endocytosis. Journal of Cell Biology. 180 (5), 905-914 (2008).
  47. Davenport, N. R., et al. Membrane dynamics during cellular wound repair. Molecular Biology of the Cell. 27 (14), 2272-2285 (2016).
  48. Defour, A., Sreetama, S. C., Jaiswal, J. K. Imaging cell membrane injury and subcellular processes involved in repair. Journal of Visualized Experiments. (85), (2014).
  49. Lee, J. J. A., et al. Cell Membrane Repair Assay Using a Two-photon Laser Microscope. Journal of Visualized Experiments. (131), (2018).
  50. Weisleder, N., et al. Visualization of MG53-mediated cell membrane repair using in vivo and in vitro systems. Journal of Visualized Experiments. (52), (2011).
  51. Corrotte, M., et al. Toxin pores endocytosed during plasma membrane repair traffic into the lumen of MVBs for degradation. Traffic. 13 (3), 483-494 (2012).
  52. Kuismanen, E., Saraste, J. Low temperature-induced transport blocks as tools to manipulate membrane traffic. Methods in Cell Biology. 32, 257-274 (1989).
  53. Togo, T., et al. The mechanism of facilitated cell membrane resealing. Journal of Cell Science. 112, 719-731 (1999).
  54. Johnson, S. A., et al. Temperature-dependent phase behavior and protein partitioning in giant plasma membrane vesicles. Biochimica et Biophysica Acta. 1798 (7), 1427-1435 (2010).
  55. Lam, J. G. T., et al. Host cell perforation by listeriolysin O (LLO) activates a Ca(2+)-dependent cPKC/Rac1/Arp2/3 signaling pathway that promotes Listeria monocytogenes internalization independently of membrane resealing. Molecular Biology of the Cell. 29 (3), 270-284 (2018).

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