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doi: 10.1242/10.1242/jcs.00455

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Biochemical characterization, distribution and phylogenetic analysis of Drosophila melanogaster ryanodine and IP3 receptors, and thapsigargin-sensitive Ca2+ ATPase

Olivia Vázquez-Martínez1, Rafael Cañedo-Merino1, Mauricio Díaz-Muñoz1,* and Juan R. Riesgo-Escovar2,*

1 Department of Molecular and Cellular Neurobiology, Neurobiology Institute, Campus UNAM-Juriquilla, Universidad Nacional Autónoma de México, Querétaro 76230, Mexico
2 Department of Developmental Neurobiology and Neurophysiology, Neurobiology Institute, Campus UNAM-Juriquilla, Universidad Nacional Autónoma de México, Querétaro 76230, Mexico



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  		Fig. 1. Scatchard analysis, pharmacological profile and Ca2+-dependence of [3H]-ryanodine binding to Drosophila melanogaster microsomal membrane fractions. Experiments were performed with 100 µg of microsomal protein and the presence of 1-120 nM (A) or 5 nM [3H]-ryanodine (B,C) as described in Materials and Methods. Panel A illustrates a hyperbolic saturation curve as a function of increasing ryanodine concentrations. The inset shows a linear Scatchard plot. This is a representative experiment from a total of 5. Panel B depicts the effects of several activators and inhibitors of RyR in comparison with control conditions (column a). column b, AMP-PCP 2 mM; column c, 10 mM MgCl2; column d, 5 µM Ruthenium Red; column e, 10 µM xanthine; column f, 2 µM dantrolene; column g, 10 nM free Ca2+; column h, the addition of 5 mM caffeine. Panel C represents the [3H]-ryanodine binding to Drosophila microsomal membranes as a function of increasing Ca2+ concentrations. The free concentrations of the cation (100 nM to 10 mM) was adjusted using EGTA and according to the Chelator program (Tatusova and Madden, 1999Go). The results in B and C are expressed as mean±s.e.m. of five independent experimental observations; where not shown, errors bars are smaller than symbols.

 


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  		Fig. 2. Scatchard analysis and pharmacological profile of [3H]-IP3 binding to Drosophila melanogaster microsomal membrane fractions. Experiments were performed with 100 µg of microsomal protein and the presence of 1-120 nM (A) or 3 nM [3H]-IP3 (B) as described in Materials and Methods. Panel A illustrates a hyperbolic saturation curve as a function of increasing IP3 concentrations. The inset shows a linear Scatchard plot. This is a representative experiment from a total of 5. Panel B depicts the inhibitory effect of 1 mg/ml heparin, 75 µM 2-APB and 5 µM xestospongin C on [3H]-IP3 binding. The results in Panel B are expressed as mean±s.e.m. of at least four independent experimental observations.

 


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  		Fig. 3. Ca2+ and Mg2+-ATPase activities in Drosophila melanogaster microsomal membrane fractions: thapsigargin sensitivity. Experiments were done with 1 µg of microsomal protein as outlined in Materials and Methods. Panel A indicates the activities of Mg2+ (filled) and Ca2+-ATPases (crosshatched). Different ATPases activities were obtained in the WT and YW stocks used. The results are expressed as mean±s.e.m. of five independent experimental observations. Panel B shows the inhibition of the Ca2+-ATPase activity promoted by increasing concentrations of thapsigargin. The results are the mean of 5 independent experimental observations. Standard errors are not shown, but in all cases were smaller to 15% of the mean value. The IC50 obtained for thapsigargin was 80 µM.

 


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  		Fig. 4. Protein localization of the ryanodine receptor, IP3 receptor and thapsigargin-sensitive Ca2+-ATPase in Drosophila melanogaster embryos. Embryos were processed as mentioned in Materials and Methods, and then incubated with TX-R-BODIPY-ryanodine (1 µM), FL-Heparin (2 µM) and FL-BODIPY-thapsigargin (5 µM). Panels A, B and C show the profuse signal elicited by TX-R-BODIPY-ryanodine (stage 5 embryo), FL-Heparin (stage 8 embryo), and FL-BODIPY-thapsigargin (stage 5 embryo), respectively. Drosophila embryos are approximately 500 µm long. In all embryo panels, anterior is left, and dorsal is top. Panels A', B' and C' show the signal elicited in late embryos (stages 15-17) incubated with TX-R-BODIPY-ryanodine (1 µM), FL-Heparin (2 µM) and FL-BODIPY-thapsigargin (5 µM), respectively. To demonstrate the specificity of the fluorescent ligands, panels A", B" and C" depict similar embryos but with a very decreased signal as a consequence of a pretreatment with high concentrations of ryanodine (80 µM), heparin (100 µM), and thapsigargin (120 µM), respectively. Panel D shows a 10x magnification of the area marked between the two white arrows in A, where it is possible to see the cytoplasmic localization of the signal (red arrow). Panel E illustrates a 20x magnification of the area marked between the white arrows in B, where the cytoplasmic nature of the labeling of heparin (2 µM) is clearly seen. Panel F shows a 1000x magnification of cells of an early embryo (stage 6) where the cytoplasmic labeling of FL-BODIPY-thapsigargin (5 µM) is seen. Panel G shows dual labeling of TX-R-BODIPY-ryanodine with FL-Heparin of the embryo shown in A' and C', and panel H shows dual labeling of TX-R-BODIPY-ryanodine with FL-BODIPY-thapsigargin of the embryo shown in B'. In these last two panels colocalization of signals is shown as yellow. These images are representative examples of more than 20 independent experiments.

 


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  		Fig. 5. Protein localization of ryanodine receptor, IP3 receptor and thapsigargin-sensitive Ca2+-ATPase in Drosophila melanogaster adults. Cryostat sections were processed as mentioned in Materials and Methods, and then incubated with TX-R-BODIPY-ryanodine (1 µM), FL-Heparin (2 µM) and FL-BODIPY-thapsigargin (5 µM). Panel A shows a sagital section of an adult male fly incubated with TX-R-BODIPY-ryanodine. Staining is seen in many tissues derived from all three germinal layers. Panel B is the same section as in A, but showing the signal elicited with FL-Heparin staining; profuse labeling is also seen, as in A. In both panels, intense labeling is seen in muscle (marked fm and lm in A) and in the intestine (marked i in A). Adult male flies are approximately 2-3 mm long. Panel C shows two images from the same section at 100x magnification. The image on the left shows labeling of TX-R-BODIPY-ryanodine (red) and the image on the right shows FL-BODIPY-thapsigargin (green) staining of a horizontal section of the retina and optic lobe. Intense colocalization of staining is seen in the neuropils of the optic lobe [lamina (l), medulla (m), lobula and lobula plate (lp)], with lesser staining in the photoreceptor cells and optic lobe neuronal cell bodies. As shown here, colocalization of TX-R-BODIPY-ryanodine and FL-BODIPY-thapsigargin was also coincidental in all adult tissues examined. Panel D shows a 1000x magnification of indirect flight muscles stained with TX-R-BODIPY-ryanodine in longitudinal section, where the striated pattern of labeling is evident, and panel D' shows a cross-section of leg muscles also stained with TX-R-BODIPY-ryanodine. In both cases, staining is cytoplasmic. Panel E shows a 1000x magnification of intestinal cells of an adult marked with TX-R-BODIPY-ryanodine in their cytoplasm. These sections are representative of 20 independent experiments. Abbreviations: a, antenna; b, brain; fm, indirect flight muscles; g, gonad; i, intestine; l, lamina; lm, leg muscle; lp, lobula and lobula plate (in the section in C the lobula plate is directly underneath the lobula); m, medulla; p, proboscis; r, retina and photoreceptor cells.

 


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  		Fig. 6. Phylogenetic analyses of relationships based on comparisons of complete amino acid sequences representing 28 different RyRs and IP3Rs. The analyses include the RyR and IP3R from Drosophila melanogaster, the calcium release channels from other invertebrates as well as the three different vertebrate isoforms of these proteins from representative species. (A) Phylogram of the most parsimonius unrooted PHYLO_WIN tree recovered (1101 steps), where horizontal branch lengths are proportional to the amount of divergence along a lineage. The topology of this tree was identical to the Neighbor Joining tree (data not shown). In Panels A and B, RyR and IP3R are depicted as `RyR' and `InsP3' followed by the type number (for vertebrate sequences) and the abbreviation of the species, respectively. (B) Unrooted cladogram illustrating the pattern of relationships. This tree was recovered following Bootstrap analysis (2 replicates). The number of each node indicates the percentage recovery for the node during resampling. Species code: Drosophila, Dm; C. elegans, Ce; blue marlin, Mn; pig, Ss; rabbit, Oc; human, Hs; mouse, Mm; bull frog, Rc; chicken, Gg; american mink, Mv; lobster, Pa; Wistar rat, Rn; cow, Bt; Xenopus, Xl.

 


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  		Fig. 7. Phylogenetic analyses of relationships based on comparisons of complete amino acid sequences representing 21 different sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs). The analyses include the SERCA from Drosophila melanogaster, the SERCAs from other invertebrates as well as the three different vertebrate isoforms from representative species. (A) Phylogram of the most parsimonius unrooted PHYLO_WIN tree recovered (1369 steps), where horizontal branch lengths are proportional to the amount of divergence along a lineage. The topology of this tree was identical to the Neighbor Joining tree (data not shown). In Panels A and B SERCAs are depicted as `ATA' followed by the type number (for vertebrate sequences) and the abbreviation of the species. (B) Unrooted cladogram illustrating the pattern of relationships. This tree was recovered following Bootstrap analysis (5 replicates). The number of each node indicates the percentage recovery for the node during resampling. Species code: Drosophila, Dm; blood fluke, Sm; yesso scallop, Py; crayfish, Pc; human, Hs; dog, Cf; cat, Fc; chicken, Gg; blue marlin, Mn; mouse, Mm; rabbit, Oc; edible frog, Re; Wistar rat, Rn; pig, Ss.

 

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© The Company of Biologists Ltd 2003