Abstract
The introduction of microinjection of trace amounts of a fluorescently labeled protein (1,2) into cultured cells leads to its incorporation into the cell’s pool of endogenous protein. Provided the microinjected protein has retained the properties of the native protein, it will become incorporated into the same structures as the endogenous protein and will serve, therefore, as a marker of the native protein’s distribution in the cell. This allows the changes in a protein’s localization to be followed in live cells in response to normal functions such as movement (3) or division (4) or formation of structures like stress fibers or myofibrils (5–9). Responses of the protein to inhibitors of cell function (11,12), or to interactions of the injected cell with other cells such as bacteria, can also be analyzed within a single live cell (13).
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References
Kreis, T. E. and Birchmeir, W. (1982) Microinjection of fluorescently labeled proteins into living cells with emphasis on cytoskeletal proteins. Int. Rev. Cytol. 75, 209–228.
Feramisco, J. R. (1979) Microinjection of fluorescently labeled alpha-actinin into living fibroblasts. Proc. Natl. Acad. Sci. USA 76, 3967–3971.
Taylor, D. L., Amato, P. A., Luby-Phelps, K., and McNeil, P. (1984) Fluorescent analog cytochemistry. Trends Biochem. Sci. 9, 88–91.
Salmon, E. D., Leslie, R. J., Saxton, W. M., Karow, M. L., and McIntosh, J. R. (1984) Spindle dynamics in sea urchin embryos: analysis using a fluorescent labeled tubulin and measurements of fluorescence redistribution after laser photobleaching. J. Cell Biol. 99, 2165–2174.
Danowski, B. A. (1998) Microtubule dynamics in serum-starved and serum-stimulated Swiss 3T3 mouse fibroblasts: implications for the relationship between serum-induced contractility and microtubules. Cell Motil. Cytoskel. 40, 1–12.
Mittal, B., Sanger, J. M., and Sanger, J. W. (1987) Visualization of myosin in living cells. J. Cell Biol. 105, 1753–1760.
Sanger, J. M., Mittal, B., Dome, J. S., and Sanger, J. W. (1989) Analysis of cell division using fluorescently labeled actin and myosin in living PtK2 cells. Cell Motil. Cytoskel. 14, 201–219.
Wang, Y.-L. (1984) Reorganization of actin filament bundles in living fibroblasts. J. Cell Biol. 99, 1478–1485.
Sanger, J. M., Mittal, B., Pochapin, M. B., and Sanger, J. W. (1986) Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin. J. Cell Biol. 102, 2053–2066.
Sanger, J. M., Pochapin, M. B., Mittal, B., and Sanger, J. W. (1987) Stress fiber and cleavage furrow formation in living cells microinjected with fluorescently labeled alphaactinin. Cell Motil. Cytoskel. 7, 209–220.
Sanger, J. M., Dabiri, G., Mittal, B., Kowalski, M. A., Haddad, J. G., and Sanger, J. W. (1990) Disruption of microfilament organization in living non-muscle cells by microinjection of Vitamin-D binding protein or DNAse I. Proc. Natl. Acad. Sci. USA 87, 5474–5478.
Dabiri, G., Sanger, J. M., Portnoy, D., and Southwick, F. (1990) Listeria monocytogenes moves rapidly through the host cell cytoplasm by inducing directional actin assembly. Proc. Natl. Acad. Sci. USA 87, 6068–6072.
Pavalko, F. M. and Burridge, K. (1991) Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin. J. Cell Biol. 114, 481–491.
Masaki, T., Endo, M., and Ebashi, S. (1968) Localization of 6S component of alphaactinin at Z-Bands. J. Biochem. (Tokyo, Japan) 62, 630–632.
Lazarides, E. and Burridge, K. (1975) Alpha-actinin: immunofluorescent localization of a muscle structural protein in non-muscle cells. Cell 6, 289–298.
Sanger, J. W., Sanger, J. M., and Jockusch, B. M. (1983) Differences in the stress fibers between fibroblasts and epithelial cells. J. Cell Biol. 96, 961–969.
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.
Feramisco, J. R. and Burridge, K. (1980) A rapid purification of alpha-actinin, filamin and a 130,000 dalton protein from smooth muscle. J. Biol. Chem. 255, 1194–1199.
McKenna, N., Meigs, J. B., and Wang, Y.-L. (1985) Exchangeability of alpha-actinin in living cardiac fibroblasts and muscle cells. J. Cell Biol. 103, 2163–2171.
Sanger, J. W., Mittal, B., and Sanger, J. M. (1984) Interaction of fluorescently labeled contractile proteins with the cytoskeleton in cell models. J. Cell Biol. 99, 918–928.
Sanger, J. M., Dome, J. S., Mittal, B., Somlyo, A. V., and Sanger, J. W. (1989) Dynamics of the endoplasmic reticulum in living non-muscle and muscle cells. Cell Motil. Cytoskel. 13, 301–319.
Waterman-Storer, C., Sanger, J. W., and Sanger, J. M. (1993) Dynamics of organelles in the mitotic spindle of living cells: membrane microtubule interactions. Cell Motil. Cytoskel. 26, 19–39.
Sanger, J. M., Pochapin, M. B., and Sanger, J. W. (1985) Midbody sealing after cytokinesis in embryos of the sea urchin Arbacia punctulata. Cell Tissue Res. 240, 287–292.
Bartoli, M. and Claycomb, W. C. (1997) Transfer of macromolecules into living adult cardiomyocytes by microinjection. Mol. Cell. Biochem. 172, 103–109.
LoRusso, S. M., Imanaka-Yoshida, K., Shuman, H., Sanger, J. M., and Sanger, J. W. (1992) Incorporation of fluorescently labeled contractile proteins into freshly isolated living adult cardiac myocytes. Cell Motil. Cytoskel. 21, 111–122.
Rhee, D., Sanger, J. M., and Sanger, J. W. (1994) The premyofibril: evidence for its role in myofibrillogenesis. Cell Motil. Cytol. 28, 1–24.
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© 2000 Humana Press Inc., Totowa, NJ
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Sanger, J.M., Danowski, B.A., Sanger, J.W. (2000). Microinjection of Fluorescently Labeled α-Actinin into Living Cells. In: Tuan, R.S., Lo, C.W. (eds) Developmental Biology Protocols. Methods in Molecular Biology™, vol 137. Humana Press. https://doi.org/10.1385/1-59259-066-7:449
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DOI: https://doi.org/10.1385/1-59259-066-7:449
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