Expression of membrane targeted aequorin in Xenopus laevis oocytes.

We described here a system for high level of expression of the calcium activated photoprotein aequorin. This protein has been targeted to the plasma membrane of Xenopus oocyte by nuclear microinjection of a plasmid containing a construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and the 5-HT1A receptor. The expression of this fusion protein is placed under the control of RSV promoter. Functional photoprotein was reconstituted in the oocyte by incubation with coelenterazine. The amount of photoprotein 24 h after nuclear microinjection of the plasmid was sufficient to trigger a detectable light emission following calcium entry. The efficiency of the expression is correlated with the dose of plasmid injected. Intracytoplasmic injection of the plasmid always failed in photoprotein expression. Targeting of the apoprotein was demonstrated by immunolocalization under confocal microscopy. In our experimental conditions, the apoprotein was always localized at the animal pole above the nucleus. We never observed expression and targeting to the plasma membrane of the vegetal pole. WE suggest that such expression might be of great interest for the study of numerous problems of developmental biology, in which calcium-dependent pathways are involved.

[1]  A. Duprat,et al.  In vivo labelling of L-type Ca2+ channels by fluorescent dihydropyridine: correlation between ontogenesis of the channels and the acquisition of neural competence in ecotderm cells from Pleurodeles waltl embryos. , 1995, Cell calcium.

[2]  A. Campbell,et al.  Nucleoplasmin-targeted aequorin provides evidence for a nuclear calcium barrier. , 1995, Experimental cell research.

[3]  A. Duprat,et al.  Increased internal Ca2+ mediates neural induction in the amphibian embryo. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[4]  R. Deguchi,et al.  Meiosis reinitiation from the first prophase is dependent on the levels of intracellular Ca2+ and pH in oocytes of the bivalves Mactra chinensis and Limaria hakodatensis. , 1994, Developmental biology.

[5]  P. Hepler,et al.  The role of calcium in cell division. , 1994, Cell calcium.

[6]  T. Pozzan,et al.  Nuclear Ca2+ concentration measured with specifically targeted recombinant aequorin. , 1993, The EMBO journal.

[7]  P. Guerrier,et al.  Evidence for the involvement of internal calcium stores during serotonin-induced meiosis reinitation in oocytes of the bivalve mollusc Ruditapes philippinarum. , 1993, Developmental biology.

[8]  P. Cochard,et al.  Lineage analysis of early neural plate cells: cells with purely neuronal fate coexist with bipotential neuroglial progenitors. , 1993, Developmental biology.

[9]  J. Kendall,et al.  Targeting aequorin to the endoplasmic reticulum of living cells. , 1992, Biochemical and biophysical research communications.

[10]  M. Lane,et al.  Promoter-cDNA-directed heterologous protein expression in Xenopus laevis oocytes. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[11]  T. Pozzan,et al.  Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin , 1992, Nature.

[12]  T. Sejnowski,et al.  Calcium-induced release of calcium regulates differentiation of cultured spinal neurons , 1991, Neuron.

[13]  O. Shimomura.  Preparation and handling of aequorin solutions for the measurement of cellular Ca2+. , 1991, Cell calcium.

[14]  J. R. Blinks.  Use of calcium-regulated photoproteins as intracellular Ca2+ indicators. , 1989, Methods in enzymology.

[15]  Y. Oron,et al.  Differences in receptor-evoked membrane electrical responses in native and mRNA-injected Xenopus oocytes. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[16]  A. Campbell.  Chemiluminescence : principles and applications in biology and medicine , 1988 .

[17]  Y. Sakaki,et al.  Site-specific mutagenesis of the calcium-binding photoprotein aequorin. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Y Sakaki,et al.  Cloning and sequence analysis of cDNA for the luminescent protein aequorin. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[19]  M. J. Cormier,et al.  Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein. , 1985, Biochemical and biophysical research communications.

[20]  L. Jaffe.  4 – The Role of Calcium Explosions, Waves, and Pulses in Activating Eggs , 1985 .

[21]  J. Vilain,et al.  U.V. Irradiation Inhibits The Electrical Block to Polyspermy in Echinoderms * , 1985, Development, growth & differentiation.

[22]  P. Andreuccetti,et al.  The modifications of cortical endoplasmic reticulum during in vitro maturation of Xenopus laevis oocytes and its involvement in cortical granule exocytosis. , 1984, The Journal of experimental zoology.

[23]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[24]  M. Wickens,et al.  The use of Xenopus oocytes for the expression of cloned genes. , 1983, Methods in enzymology.

[25]  G. Malacinski,et al.  Axial structure development in ultraviolet-irradiated (notochord-defective) amphibian embryos. , 1981, Developmental biology.

[26]  R. Elinson.  The Amphibian Egg Cortex in Fertilization and Early Development , 1980 .

[27]  R. Wallace,et al.  Protein incorporation by isolated amphibian oocytes III. Optimum incubation conditions , 1973 .