利用光轉換變色螢光蛋白Kaede在活體果蠅腦中監測週期性蛋白質合成

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

鄧淑方(Shu-Fang Teng)

指導教授 ： 江安世

國立清華大學/生命科學院/生物科技研究所/碩士(2006年)

摘要

果蠅體內有一群時鐘蛋白質於一天內會有週期性的合成與降解，藉由這些蛋白質量多寡的變化來調控果蠅日週期性的活動量與行為，而這些蛋白質包含PERIOD (簡稱PER)、 TIMELESS(簡稱TIM)與色素分散因子(Pigment-dispersing factor, 簡稱PDF)。 PDF只表現在果蠅腦中八顆調控生物時鐘的神經元 (又稱為腹側神經元, ventral Lateral neurons)，而PER與TIM則表現在上述八顆神經元及其他神經元中。到目前為止尚未清楚這些調控生物時鐘的日週期蛋白質之表現量除了在特定時鐘神經元中有週期性變化，是否在其他神經元中也有如此的變化。為了解決這個問題，我們轉殖兩株轉基因果蠅株，UAS-Kaede與UAS–nls-Kaed，此二果蠅株經由特定啟動子驅使Gal4產生以驅動Kaede表現後，可用來監測果蠅腦內神經元中蛋白質週期性的合成變化。Kaede是一種可以被光激發而轉換顏色的螢光蛋白，一般情況下Kaede散發明亮的綠色螢光，當受到紫外光照射之後，Kaede迅速地轉變成明亮且穩定的紅色螢光。我首先利用elav-Gal4與pdf-Gal4驅動Kaede表現在果蠅腦中所有的神經元以及時鐘神經元，並證實在活體果蠅腦中Kaede仍保有依光轉換顏色的特性。Kaede之合成可被蛋白質合成抑制劑cycloheximide所抑制且依劑量不同而有不同的抑制效果。而依上述特性使我們能夠去鑑定經歷日週期蛋白質合成變化的個別腦神經元。我們的實驗數據顯示此八顆腹側神經元都有週期性PDF、PER以及TIM的蛋白質合成。當利用per-Gal4與tim-Gal4驅動Kaede表現，意外的發現在一群靠近觸角突(antennal lobe)的神經元以及橢圓球體(ellipsoid body) 的神經元中，PER與TIM也有週期性的蛋白質合成。此結果意味著在果蠅腦中可能不止八顆時鐘腹側神經元參與調控日週期，而這些同樣有PER與TIM週期性蛋白質合成的神經元也可能參與調控果蠅的日週期。藉由Kaede的應用，可以進一步分析腦內其他有關蛋白質合成的相關功能，譬如細胞分裂或是長期記憶的形成。

關鍵字

果蠅 ； 楓葉蛋白 ； 光轉換 ； 日週期 ； 時鐘神經元 ； 時鐘基因 ； 時鐘蛋白質

並列摘要

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.

並列關鍵字

Drosophila ； Kaede ； photoconversion ； circadian ； clock neuron ； clock gene ； clock protein

參考文獻

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