The severely reduced flight ability (measured as flight time in a single flight assay on tethered flies) characteristic for Itp-r83Awc361/Itp-r83Aka1091 heterozygous flies is significantly ameliorated by Scer\GAL4elav-C155-driven expression of RalaScer\UAS.cRa in the mutant background.
Expression of Ilp2Scer\UAS.cBa in the insulin-producing cells under the control of Scer\GAL4Ilp2.PR rescues the starvation resistance and mobilization of TAGs from abdominal fat body cells seen in itgr[ku] (Itp-r83Aka1091/Itp-r83Aug3) mutant flies to wild type levels. The increase in body weight and stored TAG levels are only partially rescued.
Expression of Ilp2Scer\UAS.cBa in the insulin-producing cells under the control of Scer\GAL4Ilp2.PR partially restores the profile of TAGs and their constituent fatty acids in Itp-r83Aka1091/Itp-r83Aug3 mutants to that of the wild type under starvation.
Expression of Pi3K92EScer\UAS.T:Hsap\MYC,T:Hsap\CAAX in the fat body under the control of Scer\GAL4c729 partially rescues the starvation resistance phenotype seen in itgr[ku] (Itp-r83Aka1091/Itp-r83Aug3) mutant flies. Expression of Pi3K92EScer\UAS.T:Hsap\MYC,T:Hsap\CAAX leads to reduced levels of stored TAGs and their mobilization upon starvation as compared to itgr[ku] alone. The increase in body weight is only partially rescued.
Expression of StimScer\UAS.cAa driven by either Scer\GAL4Ilp2.PR or Scer\GAL4Ddc.PL suppresses the altered wing posture of Itp-r83Aka1091/Itp-r83Aug3 flies as well as the spontaneous hyperactivity of the dorsal longitudinal muscles. The flight ability and sustained air-puff-induced rhythmic flight patterns are also restored in these flies.
Pan-neuronal expression of StimScer\UAS.cAa and olf186-FScer\UAS.cZa driven by Scer\GAL4elav-C155 in Itp-r83Aka1091/Itp-r83Aug3 flies suppresses the abnormal wing-posture to varying extents but does not fully restore flight ability.
Expression of olf186-FScer\UAS.cZa in Ilp2 neurons (under the control of Scer\GAL4Ilp2.PR) partially suppresses the altered wing posture of Itp-r83Aka1091/Itp-r83Aug3 mutants. Although flight ability is not restored, flight patterns are initiated on air-puff delivery, while these are normally completely lacking in Itp-r83Aka1091/Itp-r83Aug3 mutants. Spontaneous hyperactivity of the DLMs in Itp-r83Aka1091/Itp-r83Aug3 mutants is suppressed to a significant extent by expression of olf186-FScer\UAS.cZa either ubiquitously (Scer\GAL4hs.PB at 25[o]C) or in the aminergic (Scer\GAL4Ilp2.PR) and glutamatergic subneuronal domains (Scer\GAL4OK371).
Ubiquitous expression of olf186-FScer\UAS.cZa under the control of Scer\GAL4hs.PB in Itp-r83Aka1091/Itp-r83Aug3 mutants, up-regulated by a heat shock either in 24hr pupae or in 1-day-old adults results in a significant number of flies initiating flight in response to an air puff. Thus, levels of olf186-FScer\UAS.cZa can modulate flight circuit activity both during its development and in adult function. However, the flight patterns obtained are not sustained and appear arrhythmic, indicating that although olf186-FScer\UAS.cZa overexpression can suppress the flight defects and associated physiology of Itp-r83Aka1091/Itp-r83Aug3 mutants to a significant extent, it is insufficient to regain complete flight.
Pan-neural overexpression of olf186-FScer\UAS.cZa in Itp-r83Aka1091/Itp-r83Aug3 neurons restores detectable SOCE to 70%, as in wild-type. Also, '[Ca[2+]'][[ER]] is restored to wild-type levels, indicating that reduced SOCE and elevated '[Ca[2+]'][[ER]] in Itp-r83Aka1091/Itp-r83Aug3 are linked homeostatic processes.
Overexpression of olf186-FScer\UAS.cZa in wild-type neurons (under the control of Scer\GAL4elav-C155) does not afffect SOCE and '[Ca[2+]'][[ER]], although it does elevate '[Ca[2+]'][[i]] to ~1υM in wild-type and Itp-r83Aka1091/Itp-r83Aug3 backgrounds.
Introduction of a single copy of olf186-Fk11505 suppresses the cold-sensitive lethality found in Itp-r83Aka1091/Itp-r83Aug3 mutants at 17.5[o]C. The wing posture defect found in these mutants when grown at 25[o]C is also suppressed to a significant extent.
Ca-P60AKum170/olf186-FEY01467; Itp-r83Aka1091/Itp-r83Aug3 flies exhibit normal wings and normal levels of spontaneous electrical activity in DLM recordings. Flight ability is restored in a significant number of these triple mutant flies. This is in contrast to the complete loss of flight ability in Itp-r83Aka1091/Itp-r83Aug3 mutants and in olf186-FEY01467/+; Itp-r83Aka1091/Itp-r83Aug3 or Ca-P60AKum170/+; Itp-r83Aka1091/Itp-r83Aug3 mutants. Over 60% of olf186-Fk11505/Ca-P60AKum170; Itp-r83Aka1091/Itp-r83Aug3 adults and approximately 50% of olf186-FEY01467/Ca-P60AKum170; Itp-r83Aka1091/Itp-r83Aug3 adults pass as 'fliers' in the cylinder drop test assay. Air-puff delivery elicited sustainable rhythmic flight patterns similar to wild-type in a high proportion of these flies.
The presence of olf186-FEY01467 in either olf186-FEY01467/+; Itp-r83Aka1091/Itp-r83Aug3 or olf186-FEY01467/Ca-P60AKum170; Itp-r83Aka1091/Itp-r83Aug3 organisms restored carbachol-stimulated Ca[2+] release to wild-type levels.
The abnormal wing posture phenotype of Itp-r83Aka1091/Itp-r83Aug3 animals raised at 25'C is suppressed by Ca-P60AKum170/+. Lethality due to Itp-r83Aka1091/Itp-r83Aug3 in animals raised at 17'C is also suppressed by Ca-P60AKum170/+. The resulting animals have the same abnormal wing posture phenotype as Itp-r83Aka1091/Itp-r83Aug3 animals raised at 25'C.
The flight defects seen in Itp-r83Aka1091/Itp-r83Aug3 animals raised at 25'C as measured by the "cylinder drop assay" test are not suppressed by Ca-P60AKum170/+. However, the abnormally high frequency of spontaneous action potentials in dorsal longitudinal flight muscles of these adults and their lack of flight response to stimulation with a gentle puff of air are both significantly suppressed by Ca-P60AKum170/+.
Expression of Itp-r83AScer\UAS.cVa in the insulin-producing cells under the control of Scer\GAL4Ilp2.PR rescues the starvation resistance seen in itgr[ku] (Itp-r83Aka1091/Itp-r83Aug3) mutant flies to wild type levels. The lipid level defects in the fat body cells of starved flies is also rescued, as is the increase in body weight and TAG level. Expression in the fat body under the control of Scer\GAL4c729 partially rescues the starvation resistance phenotype, increase in body weight and abdominal TAG level.
Expression of Itp-r83AScer\UAS.cVa in the insulin-producing cells under the control of Scer\GAL4Ilp2.PR partially restores the profile of TAGs and their constituent fatty acids in Itp-r83Aka1091/Itp-r83Aug3 mutants close to that of the wild type under starvation.
Complete rescue of the altered wing posture is seen in Itp-r83AScer\UAS.cVa; Scer\GAL4Ilp2.PR/+; Itp-r83Aka1091/Itp-r83Aug3 animals. Flight ability is also rescued significantly in these flies. The delivery of an air puff stimulus to Itp-r83Aka1091/Itp-r83Aug3 flies expressing Scer\GAL4Ilp2.PR-driven Itp-r83AScer\UAS.cVa leads to rhythmic generation of action potentials similar to what is observed in wild-type flies. However, the response in some flies is more erratic and could not be maintained for more than five seconds.
Wing posture and air puff-induced flight (assessed in single fly tethered assays) are not rescued when Itp-r83AScer\UAS.cVa; Scer\GAL4Ilp2.PR/Scer\GAL80[ts.αTub84B]; Itp-r83Aka1091/Itp-r83Aug3 animals are grown at 19[o]C during pupal and adult stages, but are completely rescued when these animals are grown at 30[o]C. Itp-r83AScer\UAS.cVa; Scer\GAL4Ilp2.PR/Scer\GAL80[ts.αTub84B]; Itp-r83Aka1091/Itp-r83Aug3 organisms grown at 30[o]C during pupal stages, but shifted to 19[o]C as adults, have normal wing posture and air puff-induced flight.