Abstract
Cytoplasmic streaming in the characean algae is inhibited by micromolar rises in the level of cytosolic free Ca2+, but both the mechanism of action and the molecular components involved in this process are unknown. We have used monoclonal antibodies against soybean Ca2+-dependent protein kinase (CDPK), a kinase that is activated by micromolar Ca2+ and co-localizes with actin filaments in higher-plant cells (Putnam-Evans et al., 1989, Cell Motil. Cytoskel. 12, 12–22) to identify and localize its characean homologue. Immunoblot analysis revealed that CDPK in Chara corralina Klein ex. Wild shares the same relative molecular mass (51–55 kDa) as the kinase purified from soybean, and after electrophoresis in denaturing gels is capable of phosphorylating histone III-S in a Ca2+-dependent manner. Immunofluorescence microscopy localized CDPK in Chara to the subcortical actin bundles and the surface of small organelles and other membrane components of the streaming endoplasm. The endoplasmic sites carrying CDPK were extracted from internodal cells by vacuolar perfusion with 1 mM ATP or 10−4 M Ca2+. Both the localization of CDPK and its extraction from internodal cells by perfusion with ATP or high Ca2+ are properties similar to that reported for the heavy chain of myosin in Chara (Grolig et al., 1988, Eur. J. Cell Biol. 47, 22–31). Based on its endoplasmic location and inferred enzymatic properties, we suggest that CDPK may be a putative element of the signal-transduction pathway that mediates the rapid Ca2+-induced inhibition of streaming that occurs in the characean algae.
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Abbreviations
- CDPK:
-
calcium-dependent protein kinase
- kDa:
-
kilodalton
- mAb:
-
monoclonal antibody
- Mr :
-
relative molecular mass
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
References
Adams, R.J., Pollard, T.D. (1986) Propulsion of organelles isolated from Acanthamoeba along actin filaments by myosin-I. Nature 322, 754–756
Adams, R.J., Pollard, T.D. (1989a) Binding of myosin I to membrane lipids. Nature 340, 565–568
Adams, R.J., Pollard, T.D. (1989b) Membrane-bound myosin-I provides new mechanisms in cell motility. Cell Motil. Cytoskel. 14, 178–182
Grolig, F., Williamson, R.E., Parke, J., Miller, C., Anderton, B.H. (1988) Myosin and Ca2+-sensitive streaming in the alga Chara: two polypeptides reacting with a monoclonal anti-myosin and their localization in the streaming endoplasm. Eur. J. Cell Biol. 47, 22–31
Harmon, A.C., McCurdy, D.W. (1990) Calcium-dependent protein kinase and its possible role in the regulation of the cytoskeleton. In: Current topics in plant biochemistry and physiology, vol. 9, pp. 119–128, Randall, D.D., Blevins, D.G., eds. University of Missouri-Columbia
Harmon, A.C., Putnam-Evans, C., Cormier, M.J. (1987) A calciumdependent but calmodulin-independent protein kinase from soybean. Plant Physiol. 83, 830–837
Harper, J.F., Sussman, M.R., Schaller, G.E., Putnam-Evans, C., Charbonneau, H., Harmon, A.C. (1991) A calcium-dependent protein kinase with a regulatory domain similar to calmodulin. Science 252, 951–954
Kachar, B., Reese, T.S. (1988) The mechanism of cytoplasmic streaming in Characean algal cells: sliding of endoplasmic reticulum along actin filaments. J. Cell Biol. 106, 1545–1552
Kamiya, N. (1981) Physical and chemical basis of cytoplasmic streaming. Annu. Rev. Plant Physiol. 32, 205–236
Kamiya, N. (1986) Cytoplasmic streaming in giant algal cells: a historical survey of experimental approaches. Bot. Mag. Tokyo 69, 544–554
Korn, E.D., Hammer, J.A. III (1988) Myosins of nonmuscle cells. Annu. Rev. Biophys. Biophysic. Chem. 17, 23–45
Korn, E.D., Hammer, J.A. III (1990) Myosin I. Curr. Opin. Cell Biol. 2, 57–61
Kuznicki, J. (1986) Phosphorylation of myosin in non-muscle and smooth muscle cells. Possible rules and evolutionary trends. FEBS Lett. 204, 169–176
Lessard, J.L. (1988) Two monoclonal antibodies to actin: one muscle selective and one generally reactive. Cell Motil. Cytoskel. 10, 349–362
McCurdy, D.W., Williamson, R.E. (1991) Actin and actin-associated proteins. In: The cytoskeletal basis of plant growth and form, pp. 3–14, Lloyd, C.L., ed. Academic Press, London
Mooseker, M.S., Coleman, T.R. (1989) The 110-kD proteincalmodulin complex of the intestinal microvillus (brush border myosin I) is a mechanoenzyme. J. Cell Biol. 108, 2395–2400
Putnam-Evans, C., Harmon, A.C., Palevitz, B.A., Fechheimer, M., Cormier, M.J. (1989) Calcium-dependent protein kinase is localized with F-actin in plant cells. Cell Motil. Cytoskel. 12, 12–22
Putnam-Evans, C., Harmon, A.C., Cormier, M.J. (1990) Purification and characterization of a novel calcium-dependent protein kinase from soybean. Biochemistry 29, 2488–2495
Shimmen, T., Tazawa, M. (1983) Control of cytoplasmic streaming by ATP, Mg2+ and cytochalasin B in permeabilized Characeae cell. Protoplasma 115, 18–24
Tazawa, M., Shimmen, T. (1987) Cell motility and ionic relations in characean cells as revealed by internal perfusion and cell models. Int. Rev. Cytol. 109, 259–312
Tominaga, Y., Tazawa, M. (1981) Reversible inhibition of cytoplasmic streaming by intracellular Ca2+ in tonoplast-free cells of Chara australis. Protoplasma 109, 103–111
Tominaga, Y., Shimmen, T., Tazawa, M. (1983) Control of cytoplasmic streaming by extracellular Ca2+ in permeabilized Nitella cells. Protoplasma 116, 75–77
Tominaga, Y., Muto, S., Shimmen, T., Tazawa, M. (1985) Calmodulin and Ca2+-controlled cytoplasmic streaming in characean cells. Cell Struct. Funct. 10, 315–325
Tominaga, Y., Wayne, R., Tung, H.Y.L., Tazawa, M. (1987) Phos-phorylation-dephosphorylation is involved in Ca2+-controlled cytoplasmic streaming of Characean cells. Protoplasma 136, 161–169
Towbin, H., Staehelin, T., Gordon, T. (1979) Electrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354
Williamson, R.E. (1975) Cytoplasmic streaming in Chara: a cell model activated by ATP and inhibited by cytochalasin B. J. Cell Sci. 17, 655–668
Williamson, R.E. (1978) Cytochalasin B stabilises the subcortical actin bundles of Chara against a solution of low ionic strength. Cytobiologie 18, 107–113
Williamson, R.E. (1991) Cytoplasmic streaming in Characean algae: mechanism, regulation by Ca2+ and organization. In: Algal cell motility, pp. 73–98, Melkonian, M., ed. Chapman and Hall, London
Williamson, R.E., Ashley, C.C. (1982) Free Ca2+ and cytoplasmic streaming in the alga Chara. Nature 296, 647–651
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We thank Dr. Richard Williamson (Plant Cell Biology Group, Research School of Biological Sciences, The Australian National University) for valuable discussions during the course of this research. This work was supported by funds from a Queen Elizabeth II Fellowship awarded to D.W.McC. and U.S. Department of Agriculture (88-37261-4199) and National Research Inititive Competitive Grants Program (91-37304-6654) grants to A.C.H.
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McCurdy, D.W., Harmon, A.C. Calcium-dependent protein kinase in the green alga Chara . Planta 188, 54–61 (1992). https://doi.org/10.1007/BF00198939
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DOI: https://doi.org/10.1007/BF00198939