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Ratio confocal imaging of free cytoplasmic calcium gradients in polarising and polarised Fucus zygotes

Published online by Cambridge University Press:  26 September 2008

Frederic Berger  and
Colin Brownlee
Frederic Berger*
Affiliation:
Ecole Normale Suprieure de Lyon, France and Marine Biological Association, Plymouth, UK
Colin Brownlee*
Affiliation:
Ecole Normale Suprieure de Lyon, France and Marine Biological Association, Plymouth, UK
*
Colin Brownlee, Marine Biological Association, The Laboratory, Citadel Hill, Plymouth PL1 2PB, UK. Telephone: (0752) 222772. Fax: (0752) 226865.
Colin Brownlee, Marine Biological Association, The Laboratory, Citadel Hill, Plymouth PL1 2PB, UK. Telephone: (0752) 222772. Fax: (0752) 226865.
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Summary

In the marine brown alga, Fucus, two poles are differentiated before cell division determining the future rhizoid or thallus. We have used a combination of the Ca2+ -sensitive dye Calcium Green and the pH-sensitive dye SNARF monitored at pH-insensitive wavelengths to obtain confocal ratio images of free cytoplasmic calcium distribution at different stages in polarising Fucus zygotes. These dyes have the advantage that they can be used in most confocal microscopes and their longer excitation wavelengths greatly reduce autofluorescence problems. Dyes of varying molecular weights (free acid form, 10 000 mol.wt or 70 000 mol.wt dextran-conjugated) were pressure microinjected into early zygotes which were allowed to polarise in unidirectional light. Dextran-conjugated dyes remained non-compartmentalised and fluorescence could be monitored for up to 3 days following microinjection. Currently we have been able to detect Ca2+ gradients at the tip of the rhizoid, confirming earlier results. Localised Ca2+ elevations have also been observed at the rhizoid pole of the polarising zygote before the onset of rhizoid germination. Limitations of this technique and the significance of these Ca2+ gradients are discussed.

Keywords

Confocal microscopyratio imagingCytoplasmic Ca2+Fucus serratusPolarization
Type
Article
Information
Zygote , Volume 1 , Issue 1 , February 1993 , pp. 9 - 15
Copyright
Copyright © Cambridge University Press 1993

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References

Augustine, G.J. & Neher, E. (1992). Calcium requirements for secretion in bovine chromaffin cells. J. Physiol. (Lond.) 450, 247–271.CrossRefGoogle ScholarPubMed
Brawley, S.H., Wetherbee, R. & Quatrano, R.S. (1976). Fine-structural studies of the gametes and embryo of Fucus vesiculosus L. (Phaeophyta). II. The cytoplasm of the egg and young zygote. J. Cell Sci. 20, 255–71.CrossRefGoogle ScholarPubMed
Brawley, S.H., Quatrano, R.S. & Wetherbee, R. (1977). Fine ultrastructural studies of the gametes and embryo of Fucus vesiculosus L. (Pheaophyta). III. Cytokinesis and the multicellular embryo. J. Cell Sci. 24, 275–94.CrossRefGoogle ScholarPubMed
Brownlee, C. (1989). Visualising cytoplasmic calcium in polarising zygote and growing rhizoid of Fucus serratus. Biol. Bull. 176(S), 14–7.CrossRefGoogle ScholarPubMed
Brownlee, C. (1990). Light and development: cellular and molecular aspects of photomorphogenesis in brown algae. In: Light and Life in the Sea, ed. Herring, P.J.Campbel, A.K.Whitfield, M. & Maddock, L. pp 115–26. Cambridge: Cambridge University Press.Google Scholar
Brownlee, C. & Pulsford, A.L. (1988). Visualization of the cytoplasmic Ca2+ gradient in Fucus serratus rhizoid: correlation with cell ultrastructure and polarity. J.Cell Sci. 91, 249–56.CrossRefGoogle Scholar
Brownlee, C. & Wood, J.W. (1986). A gradient of cytoplasmic free calcium in growing rhizoid cells of Fucus serratus. Nature 320, 624–6.CrossRefGoogle Scholar
Gilroy, S. & Jones, R.L. (1992). Gibberellic acid and abscisic acid regulate cytoplasmic calcium and secretory activity in barley aleurone protoplasts. Proc. Natl. Acad. Sci. USA 89, 3591–5.CrossRefGoogle ScholarPubMed
Hurst, S.R. & Kropf, D.R. (1991). Ionic requirements for establishment of an embryonic axis in Pelvetia zygotes. Planta 185, 27–33.CrossRefGoogle ScholarPubMed
Jaffe, L.F. (1958). Tropistic responses of zygotes of the Fucaceae to polarized light. Exp. Cell Res. 15, 282–99.CrossRefGoogle ScholarPubMed
Kaila, K. & Voipio, J. (1985). A simple method for dry bevelling of micropipettes used in the construction of ion-selective electrodes. J. Physiol. (Lond.) 369, 8P.Google Scholar
Kasai, H. & Augustine, G.J. (1990). Cytosolic Ca2+ gradient triggering unidirectional fluid secretion from exocrine pancreas. Nature 348, 735–8.CrossRefGoogle ScholarPubMed
Kauss, H. (1987). Some aspects of calcium-dependent regulation in plant metabolism. Annu. Rev. Plant Physiol. 38, 47–72.CrossRefGoogle Scholar
Kropf, D.L. (1992a). Establishment and expression of cellular polarity in fucoid zygotes. Microbiol. Rev. 56, 316–39.CrossRefGoogle ScholarPubMed
Kropf, D.L. (1992 b). Role of the cytoskeleton in intracellular morphogenesis of zygotes of fucoid algae. In: The Cytoskeleton of Algae, ed. Menzel, D. pp. 79–92. Boca Raton, Florida: CRC Press.Google Scholar
Kropf, D.L., & Quatrano, R.S. (1987). Localization of membrane associated calcium during development of fucoid algae using chlorotetracycline. Planta 171, 158–70.CrossRefGoogle ScholarPubMed
Kropf, D.L., Kloareg, B. & Quatrano, R.S. (1988). Cell wall is required for fixation of the embryonic axis in Fucus zygote. Science 239, 187–90.CrossRefGoogle Scholar
Kropf, D.L., Berge, S.K. & Quatrano, R.S. (1989). Actin localization during Fucus embryogenesis. Plant Cell, 1 191–200.CrossRefGoogle ScholarPubMed
Kropf, D.L., Jordan, J.R., Victoria, W.A. & Gibbon, B.C. (1992). Cellular polarity in Pelvetia zygotes: studies in intracellular pH and division alignment. Curr. Top. Plant Biochem., Mol. Biol. Physiol. 11, (in press).Google Scholar
Kühtreiber, W.M. & Jaffe, L.F. (1990). Detection of extracellular calcium gradients with a calcium-specific vibrating electrode. J. Cell Biol. 110, 1565–73.CrossRefGoogle ScholarPubMed
Miller, D.D., Callaham, D.A., Gross, D.J. & Hepler, P.K. (1992). Free Ca2+ gradient in growing pollen tubes of Lillium. J. Cell Sci. 101, 7–12.CrossRefGoogle Scholar
Nick, P. & Furuya, M. (1992). Induction and fixation of polarity: early steps in plant morphogenesis. Dev. Growth Differ. 34, 115–25.CrossRefGoogle ScholarPubMed
Nuccitelli, R. (1978). Ooplasmic segregation and secretion in the Pelvetia egg is accompanied by a membrane-generated electrical current. Dev. Biol. 62, 13–33.CrossRefGoogle ScholarPubMed
Nuccitelli, R. & Jaffe, L.F. (1975). The pulse current pattern generated by developing fucoid eggs. J. Cell Biol. 64, 636–43.CrossRefGoogle ScholarPubMed
Quatrano, R.S. (1990). Polar axis fixation and cytoplasmic localization in Fucus. In: Genetics of Pattern Formation and Growth Control, ed. Mahowald, A., pp. 31–46. New York: A.R. Liss.Google Scholar
Rathore, K.S., Cork, R.J. & Robinson, K.R. (1991). A cytoplasmic gradient of Ca2+ is correlated with the growth of lily pollen tubes. Dev. Biol. 148, 612–19.CrossRefGoogle ScholarPubMed
Read, N.D., Allan, W.T.G., Knight, H., Knight, M.R., Mahlo, R., Russel, A., Shacklock, P.S. & Trewavas, T.J. (1992). Imaging and measurement of cytosolic free calcium in plant and fungal cells. J. Microsc. 166, 57–86.CrossRefGoogle Scholar
Robinson, K.H. & Cone, R. (1980). Polarization of fucoid eggs by a calcium ionophore gradient. Science 207, 77–8.CrossRefGoogle ScholarPubMed
Schiefelbein, J.W., Shipley, A. & Rowse, P. (1992). Calcium influx at the tip of root-hair cells of Arabidopsis thalliana. Planta 187, 455–59.CrossRefGoogle Scholar
Schroter, K. (1978). Asymmetrical jelly secretion of zygotes of Pelvetia and Fucus: an early polarization event. Planta 140, 69–73.CrossRefGoogle ScholarPubMed
Speksnijder, J.E., Miller, A.L., Weisenseel, M.H., Chen, T.H. & Jaffe, L.F. (1989). Calcium buffer injection blocks fucoid egg development by facilitating calcium diffusion. Proc. Natl. Acad. Sci USA 86, 6607–11.CrossRefGoogle ScholarPubMed
Tsien, R. Y. & Rink, T.J. (1980). Neutral carrier ion-selective microelectrodes for measurements of intracellular free calcium. Biochem. Biophys. Acta 599, 623–38.CrossRefGoogle ScholarPubMed
Vandekerckhove, J. (1990). Actin-binding proteins. Curr. Opin. Cell. Biol. 2, 41–50.CrossRefGoogle ScholarPubMed
Wagner, V.T., Brian, L. & Quatrano, R.S. (1992). Role of a vitronectin-like molecule in embryo adhesion of the brown alga Fucus. Proc. Natl. Acad. Sci USA 89, 3644–48.CrossRefGoogle ScholarPubMed
Weisenseel, M.H., Dorn, A. & Jaffe, L.F. (1979). Natural H+ currents traverse growing roots and root hairs of barley (Hordeum vulgare L.). Plant. Physiol. 64, 512–18.CrossRefGoogle ScholarPubMed