Advertisement

Cell Fusion pp 347-361 | Cite as

Quantitative Assays for Cell Fusion

  • Jessica H. Shinn-Thomas
  • Victoria L. Scranton
  • William A. Mohler
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 475)

Summary

Cell fusion would seem to be obviously recognizable upon visual inspection, and many studies employ a simple microscopic fusion index to quantify the rate and extent of fusion in cell culture. However, when cells are not in monolayers or when there is a large background of multinucleation through failed cytokinesis, cell–cell fusion can only be proven by mixing of cell contents. Furthermore, determination of the microscopic fusion index must generally be carried out manually, creating opportunities for unintended observer bias and limiting the numbers of cells assayed and therefore the statistical power of the assay. Strategies for making assays dependent on fusion and independent of visual observation are critical to increasing the accuracy and throughput of screens for molecules that control cell fusion. A variety of in vitro biochemical and nonbiochemical techniques have been developed to assay and monitor fusion events in cultured cells. In this chapter, we briefly discuss several in vitro fusion assays, nearly all based on systems of two components that interact to create a novel assayable signal only after cells fuse. We provide details for the use of one example of such a system, intracistronic complementation of β-galactosidase activity by mutants of Escherichia coli lacZ, which allows for either cell-by-cell microscopic assay of cell fusion or quantitative and kinetic detection of cell fusions in whole populations (1). In addition, we describe a combination of gene knock-down protocols with this assay to study factors required for myoblast fusion.

Key Words

Cell fusion lacZ β-galactosidase complementation fluorescence histochemistry protein interactions small interfering RNA 

Notes

Acknowledgments

This work was supported by grants from the Patterson Trust and the National Institutes of Health (HD43156) to W.A.M.

References

  1. 1.
    Mohler, W. A. and Blau, H. M. (1996) Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 93, 12423–12427.CrossRefPubMedGoogle Scholar
  2. 2.
    Blumenthal, R., Clague, M. J., Durell, S. R., and Epand, R. M. (2003) Membrane fusion. Chem. Rev. 103, 53–69.CrossRefPubMedGoogle Scholar
  3. 3.
    Chen, E. H. and Olson, E. N. (2005) Unveiling the mechanisms of cell–cell fusion. Science 308, 369–373.CrossRefPubMedGoogle Scholar
  4. 4.
    Jahn, R., Lang, T., and Sudhof, T. C. (2003) Membrane fusion. Cell 112, 519–533.CrossRefPubMedGoogle Scholar
  5. 5.
    Hu, C., Ahmed, M., Melia, T. J., Sollner, T. H., Mayer, T., and Rothman, J. E. (2003) Fusion of cells by flipped SNAREs. Science 300, 1745–1749.CrossRefPubMedGoogle Scholar
  6. 6.
    Giraudo, C. G., Hu, C., You, D., Slovic, A. M., Mosharov, E. V., Sulzer, D., Melia, T. J., and Rothman, J. E. (2005) SNAREs can promote complete fusion and hemifusion as alternative outcomes. J. Cell Biol. 170, 249–260.CrossRefPubMedGoogle Scholar
  7. 7.
    Podbilewicz, B., Leikina, E., Sapir, A., Valansi, C., Suissa, M., Shemer, G., and Chernomordik, L. V. (2006) The C. elegans developmental fusogen EFF-1 mediates homotypic fusion in heterologous cells and in vivo. Dev. Cell 11, 471–481.CrossRefPubMedGoogle Scholar
  8. 8.
    Morris, S. J., Sarkar, D. P., White, J. M., and Blumenthal, R. (1989) Kinetics of pH-dependent fusion between 3T3 fibroblasts expressing influenza hemagglutinin and red blood cells. Measurement by dequenching of fluorescence. J. Biol. Chem. 264, 3972–3978.PubMedGoogle Scholar
  9. 9.
    Kemble, G. W., Bodian, D. L., Rose, J., Wilson, I. A., and White, J. M. (1992) Intermonomer disulfide bonds impair the fusion activity of influenza virus hemagglutinin. J. Virol. 66, 4940–4950.PubMedGoogle Scholar
  10. 10.
    Bagai, S. and Lamb, R. A. (1996) Truncation of the COOH-terminal region of the paramyxovirus SV5 fusion protein leads to hemifusion but not complete fusion. J. Cell Biol. 135, 73–84.CrossRefPubMedGoogle Scholar
  11. 11.
    Sarkar, D. P., Morris, S. J., Eidelman, O., Zimmerberg, J., and Blumenthal, R. (1989) Initial stages of influenza hemagglutinin-induced cell fusion monitored simultaneously by two fluorescent events: cytoplasmic continuity and lipid mixing. J. Cell Biol. 109, 113–122.CrossRefPubMedGoogle Scholar
  12. 12.
    Fischer, C., Schroth-Diez, B., Herrmann, A., Garten, W., and Klenk, H. D. (1998) Acylation of the influenza hemagglutinin modulates fusion activity. Virology 248, 284–294.CrossRefPubMedGoogle Scholar
  13. 13.
    Chernomordik, L. V., Frolov, V. A., Leikina, E., Bronk, P., and Zimmerberg, J. (1998) The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation. J. Cell Biol. 140, 1369–1382.CrossRefPubMedGoogle Scholar
  14. 14.
    Keller, P. M., Person, S., and Snipes, W. (1977) A fluorescence enhancement assay of cell fusion. J. Cell Sci. 28, 167–177.PubMedGoogle Scholar
  15. 15.
    Huerta, L., Lopez-Balderas, N., Larralde, C., and Lamoyi, E. (2006) Discriminating in vitro cell fusion from cell aggregation by flow cytometry combined with fluorescence resonance energy transfer. J. Virol. Methods 138, 17–23.CrossRefPubMedGoogle Scholar
  16. 16.
    Nussbaum, O., Broder, C. C., and Berger, E. A. (1994) Fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virus–based assay quantitating cell fusion–dependent reporter gene activation. J. Virol. 68, 5411–2542.PubMedGoogle Scholar
  17. 17.
    Feng, Y., Broder, C. C., Kennedy, P. E., and Berger, E. A. (1996) HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein–coupled receptor. Science 272, 872–877.CrossRefPubMedGoogle Scholar
  18. 18.
    Okuma, K., Nakamura, M., Nakano, S., Niho, Y., and Matsuura, Y. (1999) Host range of human T-cell leukemia virus type I analyzed by a cell fusion–dependent reporter gene activation assay. Virology 254, 235–244.CrossRefPubMedGoogle Scholar
  19. 19.
    Russell, C. J., Kantor, K. L., Jardetzky, T. S., and Lamb, R. A. (2003) A dual-functional paramyxovirus F protein regulatory switch segment: activation and membrane fusion. J. Cell Biol. 163, 363–374.CrossRefPubMedGoogle Scholar
  20. 20.
    Yi, Y., Isaacs, S. N., Williams, D. A., Frank, I., Schols, D., De Clercq, E., Kolson, D. L., and Collman, R. G. (1999) Role of CXCR4 in cell–cell fusion and infection of mono-cyte-derived macrophages by primary human immunodeficiency virus type 1 (HIV-1) strains: two distinct mechanisms of HIV-1 dual tropism. J. Virol. 73, 7117–7125.PubMedGoogle Scholar
  21. 21.
    Broer, R., Boson, B., Spaan, W., Cosset, F. L., and Corver, J. (2006) Important role for the transmembrane domain of severe acute respiratory syndrome coronavirus spike protein during entry. J. Virol. 80, 1302–1310.CrossRefPubMedGoogle Scholar
  22. 22.
    He, B., McAllister, W. T., and Durbin, R. K. (1995) Phage RNA polymerase vectors that allow efficient gene expression in both prokaryotic and eukaryotic cells. Gene 164, 75–79.CrossRefPubMedGoogle Scholar
  23. 23.
    Dutch, R. E. and Lamb, R. A. (2001) Deletion of the cytoplasmic tail of the fusion protein of the paramyxovirus simian virus 5 affects fusion pore enlargement. J. Virol. 75, 5363–5369.CrossRefPubMedGoogle Scholar
  24. 24.
    Paterson, R. G., Russell, C. J., and Lamb, R. A. (2000) Fusion protein of the paramyxovirus SV5: destabilizing and stabilizing mutants of fusion activation. Virology 270, 17–30.CrossRefPubMedGoogle Scholar
  25. 25.
    Waning, D. L., Schmitt, A. P., Leser, G. P., and Lamb, R. A. (2002) Roles for the cytoplasmic tails of the fusion and hemagglutinin-neuraminidase proteins in budding of the paramyxovirus simian virus 5. J. Virol. 76, 9284–9297.CrossRefPubMedGoogle Scholar
  26. 26.
    Bradley, J., Gill, J., Bertelli, F., Letafat, S., Corbau, R., Hayter, P., Harrison, P., Tee, A., Keighley, W., Perros, M., Ciaramella, G., Sewing, A., and Williams, C. (2004) Development and automation of a 384-well cell fusion assay to identify inhibitors of CCR5/CD4-mediated HIV virus entry. J. Biomol. Screen. 9, 516–524.CrossRefPubMedGoogle Scholar
  27. 27.
    Alvarez-Dolado, M., Pardal, R., Garcia-Verdugo, J. M., Fike, J. R., Lee, H. O., Pfeffer, K., Lois, C., Morrison, S. J., and Alvarez-Buylla, A. (2003) Fusion of bone-marrow–derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 425, 968–973.CrossRefPubMedGoogle Scholar
  28. 28.
    Harris, R. G., Herzog, E. L., Bruscia, E. M., Grove, J. E., Van Arnam, J. S., and Krause, D. S. (2004) Lack of a fusion requirement for development of bone marrow–derived epithelia. Science 305, 90–93.CrossRefPubMedGoogle Scholar
  29. 29.
    Reinecke, H., Minami, E., Poppa, V., and Murry, C. E. (2004) Evidence for fusion between cardiac and skeletal muscle cells. Circ. Res. 94, e56–e60.CrossRefPubMedGoogle Scholar
  30. 30.
    Ullmann, A., Jacob, F., and Monod, J. (1967) Characterization by in vitro complementation of a peptide corresponding to an operator-proximal segment of the beta-galactosidase structural gene of Escherichia coli. J. Mol. Biol. 24, 339–343.CrossRefPubMedGoogle Scholar
  31. 31.
    Lojda, Z. (1979) Enzyme Histochemistry: A Laboratory Manual. Springer-Verlag, Berlin.Google Scholar
  32. 32.
    Rossi, F. M., Blakely, B. T., Charlton, C. A., and Blau, H. M. (2000) Monitoring protein–protein interactions in live mammalian cells by beta-galactosidase complementation. Methods Enzymol. 328, 231–251.CrossRefPubMedGoogle Scholar
  33. 33.
    Fiering, S. N., Roederer, M., Nolan, G. P., Micklem, D. R., Parks, D. R., and Herzenberg, L. A. (1991) Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs. Cytometry 12, 291–301.CrossRefPubMedGoogle Scholar
  34. 34.
    Blakely, B. T., Rossi, F. M., Tillotson, B., Palmer, M., Estelles, A., and Blau, H. M. (2000) Epidermal growth factor receptor dimerization monitored in live cells. Nat. Biotechnol. 18, 218–222.CrossRefPubMedGoogle Scholar
  35. 35.
    Mohler, W. A. (1996) Analysis of Mouse Myoblast Fusion. Stanford University, Stanford, CA.Google Scholar
  36. 36.
    Charlton, C. A., Mohler, W. A., Radice, G. L., Hynes, R. O., and Blau, H. M. (1997) Fusion competence of myoblasts rendered genetically null for N-cadherin in culture. J. Cell Biol. 138, 331–336.CrossRefPubMedGoogle Scholar
  37. 37.
    Charlton, C. A., Mohler, W. A., and Blau, H. M. (2000) Neural cell adhesion molecule (NCAM) and myoblast fusion. Dev. Biol. 221, 112–119.CrossRefPubMedGoogle Scholar
  38. 38.
    Remy, I. and Michnick, S. W. (2007) Application of protein-fragment complementation assays in cell biology. Biotechniques 42, 137, 139, 141 passim.Google Scholar
  39. 39.
    Hu, C. D., Chinenov, Y., and Kerppola, T. K. (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol. Cell 9, 789–798.CrossRefPubMedGoogle Scholar
  40. 40.
    Hu, C. D. and Kerppola, T. K. (2003) Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat. Biotechnol. 21, 539–545.CrossRefPubMedGoogle Scholar
  41. 41.
    Jach, G., Pesch, M., Richter, K., Frings, S., and Uhrig, J. F. (2006) An improved mRFP1 adds red to bimolecular fluorescence complementation. Nat. Methods 3, 597–600.CrossRefPubMedGoogle Scholar
  42. 42.
    Jacobson, R. H., Zhang, X. J., DuBose, R. F., and Matthews, B. W. (1994) Three-dimensional structure of beta-galactosidase from E. coli. Nature 369, 761–766.CrossRefPubMedGoogle Scholar
  43. 43.
    Bhat, R. A., Lahaye, T., and Panstruga, R. (2006) The visible touch: in planta visualization of protein–protein interactions by fluorophore-based methods. Plant Methods 2, 12.CrossRefPubMedGoogle Scholar
  44. 44.
    Gunther, S., Mielcarek, M., Kruger, M., and Braun, T. (2004) VITO-1 is an essential cofactor of TEF1-dependent muscle-specific gene regulation. Nucleic Acids Res. 32, 791–802.CrossRefPubMedGoogle Scholar
  45. 45.
    Galbiati, F., Volonte, D., Engelman, J. A., Scherer, P. E., and Lisanti, M. P. (1999) Targeted down-regulation of caveolin-3 is sufficient to inhibit myotube formation in differentiating C2C12 myoblasts. Transient activation of p38 mitogen-activated protein kinase is required for induction of caveolin-3 expression and subsequent myotube formation. J. Biol. Chem. 274, 30315–30321.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Jessica H. Shinn-Thomas
    • 1
  • Victoria L. Scranton
    • 1
  • William A. Mohler
    • 1
  1. 1.Department of Genetics and Developmental BiologyUniversity of Connecticut Health CenterFarmington

Personalised recommendations