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.

Return to article