探討atg8與細胞自噬作用在渦蟲再生中所扮演的角色

Study on the Role of atg8 and Autophagy in Planarian Regeneration

曾本嵂(Ben-Lyu Tseng)

指導教授 ： 朱家瑩

國立臺灣大學/生命科學院/動物學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10343

摘要

細胞自噬為一重要的細胞代謝途徑。其負責媒介溶小體內細胞質組成物的汰換。細胞自噬接受各種壓力刺激而誘發，如營養缺乏或低氧壓力。Atg8為一媒介細胞自噬活化的細胞自噬蛋白。Atg8的脂化作用可用於監控細胞自噬現象。渦蟲以其所具有的再生能力著稱，因此可作為再生研究上的模式動物。細胞自戕以及細胞增殖於渦蟲再生時均大量表現，進而將蟲體重塑為符合原有比例的小渦蟲。為了瞭解細胞自噬在渦蟲再生中扮演的角色，我們以東洋渦蟲，一種棲息於台灣的渦蟲物種作為模式，選殖了其atg8同源基因，Djatg8。序列同源分析顯示Djatg8與人類GABARAP的相似性最高。與其他的atg8 同源基因相同，Djatg8表現於渦蟲全身，包括咽部、腸以及神經系統。將螢光標示的Djatg8在於人類HeLa細胞中表現顯示其分布於哺乳類細胞內自噬體與溶小體中。我們在酵母菌中間接證實了Djatg8能夠部分參與細胞自噬現象。藉由分析Djatg8的脂化反應，我們發現渦蟲經過游離輻射照射後的確可引起細胞自噬現象，但以同樣方法分析再生以及飢餓過程中的渦蟲，並沒有觀察到明顯的差異。利用RNA干擾去除Djatg8後並不會影響渦蟲的再生。實驗結果顯示細胞自噬可能並不是渦蟲重組過程中進行分解代謝的重要途徑，但是atg8同源基因在渦蟲的高度表現表示細胞自噬在渦蟲上仍然扮演某種重要的角色。

關鍵字

渦蟲 ； 細胞自噬 ； atg8

並列摘要

Autophagy mediates the bulk turnover of cytoplasmic constituents in lysosomes, an important process for cellular metabolism. Autophagy can be induced by various stress stimuli, e.g., nutrient depletion or hypoxic stress. Upon autophagy activation, Atg8 is one of the autophagic proteins that mediate this pathway. Lipidation of Atg8 is used as a marker to monitor autophagy activity. Planarian, due to its high regenerative ability, has been used as the animal model with which to study regeneration. During regeneration, both apoptosis and cell proliferation are evoked in planarian and the separated worm is regenerated into a well-proportioned small planarian. In order to verify if autophagy participates in planarian regeneration, we cloned the atg8 homolog (Djatg8) from Dugesia japonica, which is the only available species of planarian in Taiwan. Sequence analysis of Djatg8 indicated that it highly conserves with human GABARAP (GABA receptor associated protein). Whole mount in situ hybridization of Djatg8 showed that it is expressed throughout the whole body, including pharynx and neuron system. Expression of YFP-tagged Djatg8 in HeLa cells revealed its subcellular localization at autophagosomes/autolysosmes. We also show the functional conservation of Djatg8 in yeast autophagy pathway. By detecting the lipidation of Djatg8, we found that irradiation induces autophage pathway, while there was no significant up-regulation of autophagy during regeneration or starvation process. However, RNAi of Djatg8 does not affect the regeneration of planarian. Our results suggested that autophagy is not the major anabolic process in planarian reorganization, while the ubiquitous expression of Djatg8 implied that autophagy may be involved in other critical functions of planarian.

並列關鍵字

planarian ； autophagy ； atg8

參考文獻

Alberti, A., X. Michelet, et al. (2010). "The autophagosomal protein LGG-2 acts synergistically with LGG-1 in dauer formation and longevity in C. elegans." Autophagy 6(5).

Andrade, R. M., M. Wessendarp, et al. (2006). "CD40 induces macrophage anti-Toxoplasma gondii activity by triggering autophagy-dependent fusion of pathogen-containing vacuoles and lysosomes." The Journal of clinical investigation 116(9): 2366-2377.

Baguna, J. and R. Romero (1981). "Quantitative-Analysis of Cell-Types during Growth, Degrowth and Regeneration in the Planarians Dugesia-Mediterranea and Dugesia-Tigrina." Hydrobiologia 84(Oct): 181-194.

Bowen, I. D., T. A. Ryder, et al. (1976). "Effects of Starvation on Planarian Worm Polycelis-Tenuis Iijima." Cell Tissue Res 169(2): 193-209.

Brondsted, H. V. (1969). "Planarian Regeneration 276." Pergamon,