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Regulation of longevity by Imd and Loco signaling pathways

Investigating the mechanisms of the aging process is becoming more important and indispensable to biomedical research due to aging-related human diseases and concerns for public health. In particular, the fruit fly model system, using Drosophila melanogaster, is extremely useful to studying several aspects of the aging process, because both forward and reverse genetic approaches have been shown to be efficient and successful for elucidating the primary physiological aspects of the aging process. Recent investigations of the aging process have shown that several conserved signaling pathways are related to regulation of longevity in various organisms including yeast, worms, flies and mammals. Using D. melanogaster as an aging model system, we will study longevity mechanisms with Imd and Loco signaling pathways that are known to function in immune response and blood-barrier formation, respectively, and evolutionarily conserved between insect and human.

regulation1. Fly strain and aging test. As a wild type, the lab stock strain y1w1 (Bloomington stock center) will be used for all the experiments including stress response, aging and nutritional content studies. To equalize genetic background, the flies obtained from the outside (stock centers and other labs) will be backcrossed to the y1w1 flies six times and then homogenized for six generations. Virgin flies will be collected from the bottles in which larval density is controlled in a standard cornmeal medium without live yeast. For the aging test, 200 virgin flies (20 flies per vial) will be counted and transferred to fresh standard cornmeal vials every 2 to 3 days (Fig. 1).

fig1graph

2. Stress response and nutrient assay. For the starvation test, a group of 100 flies (20 flies per vial) will be maintained in the vials containing two filters wetted with 300 ul of water at 25°C. For the oxidation test, adult flies, starved for the initial 6 hrs, will be maintained in the vials containing two filters wetted with 300 ul of 20 mM paraquat in 5% sucrose solution at 25°C. For the heat test, adult flies will be maintained in standard cornmeal vials at 37°C with 30% humidity. For nutrient, cAMP and MnSOD assays, all 20 flies will be weighed and homogenized with a specific buffer for each assay.

3. RT-PCR and iTRAQ. For RT-PCR, oligo dT-primed cDNAs will be made from 5 ug total RNA purified from adult flies, and they will be used as templates for quantitative real-time PCR. To measure phospho-peptides, iTRAQ analyses will be performed using total protein lysates purified from adult flies in CAPR proteomics facility.