Monitoring Rhythmic Protein Synthesis in Living Drosophila Brain Neurons by Photoconvertable Kaede

• Main Authors: Shu-Fang Teng, 鄧淑方
• Other Authors: Ann-Shyn Chiang
• Format: Others
• Language: en_US
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/16967640691805754604

碩士 === 國立清華大學 === 生物科技研究所 === 94 === In Drosophila, circadian locomotion behavior is controlled by the daily synthesis and degradation of clock proteins which include PERIOD (PER), TIMELESS (TIM) and Pigment-dispersing factor (PDF). In fly brains, PDF is expressed only in eight clock neurons (ventral Lateral neurons, LNvs) while PER and TIM are seen in many additional neurons. It is not clear if these circadian proteins are cycled in all neurons with their presence or only in certain clock neurons in fly brains. To answer this question, we generated a transgenic fly carrying UAS-Kaede or UAS-nls-Kaede transgene to monitor rhythmic protein synthesis in Drosophila brain neurons under the control of the promoter-driven GAL4. Kaede is a photoconvertable fluorescent protein that normally emits bright green fluorescence until it is irradiated with UV light at which point it starts to emit a bright and stable red fluorescence. I first confirmed this photoconvertable characteristic in living fly brains using elav-Gal4 and pdf-Gal4 to drive Kaede expression in all neurons and clock neurons, respectively. The synthesis of new Kaede after the conversion, which would now green fluorescence, is inhibited by cycloheximide in a dose-dependent manner. This allows the identification of individual brain neurons undergoing daily protein synthesis. Our data indicate that all eight LNvs show daily synthesis of the PDF, PER and TIM proteins. Surprisingly, by using promoter driven per-Gal4 and tim-Gal4 to express Kaede, we found that PER and TIM also underwent daily protein synthesis in some local neurons in the antennal lobe and neurons in the ellipsoid body. These results imply that in addition to the eight clock LNs, other neurons may be involved in the regulation of circadian rhythm. Applications of the Kaede reporter in dissecting other brain functions involving in new protein synthesis such as cell division or long-term memory formation are discussed.