Summary
The arrangement of cortical microtubules in tobacco protoplasts is described using the following techniques: 1. Transmission electron microscopy (TEM) of thin sections of whole protoplasts, 2. TEM of negatively stained protoplast ghosts, and 3. Indirect immunofluorescence microscopy of protoplast ghosts. Ghosts were prepared by attaching freshly isolated protoplasts to glass coverslips or formvar/carbon-coated grids with poly-L-lysine and then bursting them either osmotically or by detergent treatment in the presence of a microtubule stabilizing buffer. Osmotic bursting of protoplasts yielded large pieces of plasma membrane with attached microtubules. These preparations proved very useful for measuring the density and length of cortical microtubules. Detergent treatment dissolved the plasma membrane and altered the distribution of cortical microtubules.
Similar content being viewed by others
References
Connolly, J. A., Kalnins, V. I., Cleveland, D. W., Kirschner, M. W., 1978: Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence. J. Cell Biol.76, 781–786.
Constaeel, F., 1975: Isolation and culture of plant protoplasts. In: Plant tissue culture methods (Gamborg, O. L., Wetter, L. R., eds.), pp. 11–21. Ottawa: National Research Council of Canada.
Fowke, L. C., Bech-Hansen, C. W., Constabel, F., Gamborg, O. L., 1974: A comparative study on the ultrastructure of cultured cells and protoplasts of soybean during cell division. Protoplasma81, 189–203.
—, 1975: Electron microscopy of protoplasts. In: Plant tissue culture methods (Gamborg, O. L., Wetter, L. R., eds.), pp. 55–62. Ottawa: National Research Council of Canada.
—,Gamborg, O. L., 1980: Applications of protoplasts to the study of plant cells. Int. Rev. Cytology68, 9–51.
Gamborg, O. L., 1975: Callus and cell culture. In: Plant tissue culture methods (Gamborg, O. L., Wetter, L. R., eds.), pp. 1–10. Ottawa: National Research Council of Canada.
Gunning, B. E. S., Hardham, A. R., Hughes, J. E., 1978: Evidence for initiation of microtubules in discrete regions of the cell cortex inAzolla root-tip cells, and an hypothesis on the development of cortical arrays of microtubules. Planta143, 161–179.
Hardham, A. R., Gunning, B. E. S., 1978: Structure of cortical microtubule arrays in plant cells. J. Cell Biol.77, 14–34.
Heath, I. B., 1974: A unified hypothesis for the role of membrane bound enzyme complexes and microtubules in plant cell wall synthesis. J. theor. Biol.48, 445–449.
Henderson, D., Weber, K., 1979: Three-dimensional organization of microfilaments and microtubules in the cytoskeleton. Immunoperoxidase labelling and stereo-electron microscopy of detergent extracted cells. Exptl. Cell Res.124, 301–316.
Hepler, P. K., Palevitz, B. A., 1974: Microtubules and microfilaments. Ann. Rev. Plant Physiol.25, 309–316.
Ledbetter, M. C., Porter, K. R., 1963: A “microtubule” in plant cell fine structure. J. Cell Biol.19, 239–250.
Lloyd, C. W., Slabas, A. R., Powell, A. J., MacDonald, G., Badley, R. A., 1979: Cytoplasmic microtubules of higher plant cells visualized with anti-tubulin antibodies. Nature279, 239–241.
Marchant, H. J., 1978: Microtubules associated with the plasma membrane isolated from protoplasts of the green algaMougeotia. Exptl. Cell Res.115, 25–30.
—,Hines, E. R., 1979: The role of microtubules and cell-wall deposition in elongation of regenerating protoplasts ofMougeotia. Planta146, 41–48.
Mazia, D., Schatten, G., Sale, W., 1975: Adhesion of cells to surfaces coated with polylysine. J. Cell Biol.66, 198–200.
Osborn, M., Weber, K., 1977 a: The detergent resistant cytoskeleton of tissue culture cells includes the nucleus and the microfilament bundles. Exptl. Cell Res.106, 339–350.
— —, 1977 b: The display of microtubules in transformed cells. Cell12, 561–571.
—,Webster, R. E., Weber, K., 1978: Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK 2 cell. J. Cell Biol.77, R 27-R 34.
Palevitz, B. A., Hepler, P. K., 1976: Cellulose microfibril orientation and cell shaping in developing guard cells ofAllium: The role of microtubules and ion accumulation. Planta132, 71–93.
Pickett-Heaps, J. D., 1974: Plant microtubules. In: Dynamic aspects of plant ultrastructure (Robards, A. W., ed.), pp. 219–255. London: McGraw-Hill.
Roberts, K., 1974: Cytoplasmic microtubules and their functions. Prog. Biophys. Mol. Biol.28, 373–420.
Robinson, D. G., 1977: Plant cell wall synthesis. In: Advances in botanical research, Vol. 5 (Woolhouse, H. W., ed.), pp. 89–151. London: Academic Press.
Valk, P. van der, Rennie, P. J., Connolly, J. A., Fowke, L. C., 1979: Techniques for studying microtubule distribution in isolated tobacco protoplasts. J. Cell Biol.83, 332 a (abstr.).
Weber, K., 1976: Visualization of tubulin-containing structures by immunofluorescence microscopy: cytoplasmic microtubules, mitotic figures and vinblastine-induced paracrystals. In: Cell Motility (Goldman, R.,Pollard, T.,Rosenbaum, J., eds.), Book A, pp. 403–417. Cold Spring Harbor Laboratory.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
van der Valk, P., Rennie, P.J., Connolly, J.A. et al. Distribution of cortical microtubules in tobacco protoplasts. An immunofluorescence microscopic and ultrastructural study. Protoplasma 105, 27–43 (1980). https://doi.org/10.1007/BF01279847
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01279847