Abstract
The introduction of the green fluorescent protein (GFP) plasmids that allow proteins and peptides to be expressed with a fluorescent tag has had a major impact on the field of cell biology. It has enabled the dynamics of a wide variety of proteins to be analyzed that could not otherwise be detected in live cells. Transient transfections of muscle and nonmuscle cells with plasmids encoding various cytoskeletal proteins ligated to green fluorescent protein or Ds red protein allow changes in the cytoskeletal network to be studied in the same cell for time periods up to several days. With this approach, proteins that could not be purified and directly labeled with fluorescent dyes and microinjected into cells can now be expressed and visualized in a wide variety of cells. Procedures are presented for transfection of the nonmuscle cell, PtK2, and primary cultures of embryonic chick myocytes, and for studying the live transfected cells.
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References
Chalfie, M., Tu, Y., Euskirchen, G. Ward, W. W., and Prasher, D. C. (1994) Green Fluorescent Protein as a marker for gene expression. Science 263, 802–805.
Prasher, D. C. (1995) Using GFP to see the light. Trends Genet. 11, 320–323.
Heim, R., Prasher, D. C., and Tsien, R. Y. (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA. 91, 12501–12504.
Heim, R., Cubitt, A. B., and R. Y. Tsien (1995) Improved green fluorescence. Nature 373, 663–664.
Yang, F., Moss, L. G., and Phillips, G. N. (1996) The molecular structure of Green Fluorescent Protein, Nature Biotechnology 14, 1246–1251.
Ormo, M., Cubitt, A. B., Kallio, K., Gross, L. A. Tsien, R. Y., and Remington, S. J. (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395.
Sanger, J. M., Danowski, B. A., and Sanger, J. W. (2000) Microinjection of fluorescently labeled alpha-actinin into living cells. In: Methods in Molecular Biology: Developmental Biology Protocols (Editors: Tuan, R. S. and Lo, C. W.), Humana Press Vol. III, 449–456.
Dabiri, G. A., Turnacioglu, K. K., Sanger, J. M., and Sanger, J. W. (1997) Myofibrillogenesis visualized in living embryonic cardiomyocytes. Proc. Natl. Acad. Sci. USA 94, 9493–9498.
Doyle, T. and Botstein, D. (1996) Movement of yeast cortical actin cytoskeleton in vivo. Proc. Natl. Acad. Sci. USA 93, 3886–3891.
Ayoob, J. C., Turnacioglu, K. K., Mittal, B., Sanger, J. M., and Sanger, J. W. (2000) Targeting Of Titin Fragments Coupled to Green Fluorescent Proteins To Z-bands In Living Cardiomyocytes. Cell Motil. Cytoskeleton 45, 67–82.
Moores, S. L., Sabry, J. H., and Spudich, J. A. (1996) Myosin dynamics in live Dictyostelium cells. Proc. Natl. Acad. Sci. USA 93, 443–446.
Gerisch, G., Albrecht, R., Heizer, C., Hodgkinson, S., and Maniak, M. (1995) Chemoattractant-controlled accumulation of coronin at the leading edge of Dictyostelium cells monitored Curr. Biol. 5, 1280–1285 (1995).
Heim, R. and Tsien, R. Y. (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence energy transfer. Curr. Biol. 6, 178–182.
Matz, M. V., Fradkov, A. F., Labas, Y. A., et al. (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nature Biotechnol 17, 969–973.
Lazarides, E. and Burridge, K. (1975) Alpha-actinin: immunofluorescent localization of a muscle structural protein in non-muscle cells. Cell 6, 289–298.
Zhukarev, V., Sanger, J. W., Sanger, J. M., Goldman, Y., and Shuman, H. (1997) Steady state fluorescence polarization analysis of rhodamine phalloidin binding to muscle. Cell Motil. Cytoskel. 37, 363–377.
Dabiri, G. A., Ayoob, J. C., Turnacioglu, K. K. Sanger, J. M., and Sanger, J. W. (1999) Use of Green Fluorescent Proteins linked to cytoskeletal proteins to analyze myofibrillogenesis in living cells. Methods in Enzymology 302, 171–186.
Weber, M., Moller, K., Welzeck, M., and Schorr, J. (1995) Effects of lipopolysaccharide on transfection efficiency in eukaryotic cells. BioTechniques 19, 930–940.
Turnacioglu, K. K., Sanger, J. W., and Sanger, J. M. (1998) Sites of monomeric actin incorporation in living PtK2 and REF-52 cells. Cell Motil. Cytoskel. 40, 59–70.
Dabiri, G. A., Turnacioglu, K. K., Ayoob, J. C., Sanger, J. M., and Sanger, J. W. (1999) Transfections of primary muscle cell cultures with plasmids coding for GFP linked to full-length and truncated muscle proteins. Methods in Cell Biology 58, 239–260.
Sanger, J. M. and Sanger, J. W. (2000) Assembly of cytoskeletal proteins into cleavage furrows of tissue culture cells. Microscopy Res. Tech. 49, 190–201.
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Ayoob, J.C., Shaner, N.C., Sanger, J.W. et al. Expression of green or red fluorescent protein (GFP or DsRed) linked proteins in nonmuscle and muscle cells. Mol Biotechnol 17, 65–71 (2001). https://doi.org/10.1385/MB:17:1:65
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DOI: https://doi.org/10.1385/MB:17:1:65