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標題: | Dynamin2 中央核肌肉病變突變體在果蠅肌肉造成之病徵分析及潛在抑制者篩檢 Phenotypic analysis of centronuclear myopathy-associated dynamin2 mutations in Drosophila |
作者: | Yu-Han Chin 金鈺頷 |
指導教授: | 劉雅雯(Ya-Wen Liu) |
共同指導教授: | 吳君泰(June-Tai Wu) |
關鍵字: | 中央核肌肉病變,肌肉,果蠅, dynamin2,centronuclear myopathy,Drosophila,t-tubule, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | Dynamin2 屬於 large GTPase,參與胞膜運送及細胞骨架形成,並透過分裂胞膜協助細胞進行胞膜的重組。哺乳類的 dynamin2 分佈全身,但其突變會造成特定組織的先天性病變,包括 G537C 突變造成的週邊神經病變,Charcot–Marie-Tooth neuropathy (CMT);以及 R465W, A618T, S619L 突變造成的骨骼肌病變,Centronuclear myopathy (CNM)。兩種病變的致病機轉仍未被透徹研究。本研究利用果蠅 (Drosophila melanogaster) 做為模式生物,觀察在肌肉中表現人類 dynamin2 時,對肌肉的功能及形態造成何種影響。我們發現表現 WT 及 CNM dynamin2 突變時,三齡幼蟲的爬行能力降低,並在成蛹之後死亡。此外在這些組別中,三齡幼蟲出現肌肉萎縮以及肌肉細胞膜特化結構 t-tubule 斷裂等現象,但收縮最小工作單位 sarcomere 結構並未受到影響。為了篩檢潛在的抑制者,我們在肌肉中進行 phosphoinositide kinases knockdown和 Amphiphysin overexpression,但並未成功抑制 dynamin2 造成的影響。另外我們也發現到 dynamin2 出現在神經肌肉接合處後突觸部分。雖然神經肌肉接合處並未產生明顯的形態改變,但在 A618T 突變中,肌肉細胞膜特化而形成的 subsynaptic reticulum (SSR) 較常態更為擴散,暗示 dynamin2可能對神經肌肉接合處後突觸部分造成直接或間接的影響。我們的研究顯示,dynamin2 造成的 t-tubule 缺陷是肌肉病變的主要成因。 Dynamin2 (Dnm2) is a large GTPase which regulates membrane trafficking and actin cytoskeleton in cells. Through its membrane fission and actin reorganization abilities, Dnm2 functions as a membrane remodeling enzyme. Despite its ubiquitous expression in mammals, the mutations of Dnm2 lead to two tissue specific, human congenital diseases: Charcot–Marie-Tooth neuropathy (CMT) and Centronuclear Myopathy (CNM). The pathogenic mechanisms of Dnm2-related CNM and CMT are not completely understood. To explore the underpinning of Dnm2-associated CNM, we utilized Drosophila melanogaster as model organism to specifically express human Dnm2 disease alleles in muscle and observed the muscle function and morphology. We found that the expression of human WT and CNM mutants Dnm2 would cause defective locomotion in larvae and subsequent lethality in pupae. Strikingly, we detected muscle atrophy and t-tubule fragmentation in Drosophila body wall muscle in CNM-associated mutations, but no effect on the sarcomere structure. To build up potential therapeutic strategy, we screened for rescuers through knockdown of phosphoinositide kinases and overexpression of Amphiphysin in muscle. We did not observe suppression effect on CNM mutations Dnm2 flies in our screening. In contrast, we found the inhibition of Phosphoinositide-3-kinase or t-tubule overproduction would deteriorate the phenotype. Furthermore, we observed Dnm2 enriched in post-synaptic NMJ. Although the NMJ morphology was not completely disrupted, the subsynaptic reticulum (SSR) became diffused in CNM-Dnm2, but CMT-Dnm2 mutants. Together, our findings suggest that t-tubule disorganization is the foundation of Dnm2-assciated CNM, and Dnm2 plays multiple roles in the muscle. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52957 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子醫學研究所 |
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