果蠅ECM蛋白具有鐵硫簇結合結構(CDGSH)的蛋白;在其序列的C端具有一個鐵硫簇結合結構。本研究發現，ECM會朝同一方向地坐落在粒線體上。當在果蠅細胞(S2 cell)與人類細胞(HeLa cell)過量表現時，會造成粒線體有膨大且聚集的現象(Enlarged and Clustering Mitochondria); 藉由其所造成的所造成的現象，所以我們將之命名為ECM。我們先將ECM與老鼠同源蛋白依照其氨基酸序列的相似處與差異處區分出四個結構區域：N端、穿膜蛋白區域、連接區域與鐵硫簇結合區域。在果蠅細胞與人類細胞中進行蛋白結構區域置換時發現，ECM的N端具有造成粒線體膨大與聚集的功能。在人類細胞中，ECM與 小鼠的同源蛋白ECM-MH-1坐落在粒線體上，而另一同源蛋白ECM-MH-2 坐落在內質網。由於ECM-MH-2坐落的位置與其他兩蛋白不同，因此我們將ECM與ECM-MH-1的穿膜蛋白區域取代ECM-MH-2的穿膜蛋白區域，並發現此區域的交換會使ECM-MH-2具有如同ECM造成粒線體膨大與聚集的功能。我們推測ECM與ECM-MH-2的N端結構對於調控粒線體膨大與聚集化現象扮演決定性的角色。在活細胞顯微影像觀察下發現，過量表現ECM確實會促進粒線體聚集，並在最後進行細胞分化; 這表示粒線體的動態變化可能受到細胞週期的調控。大量表現ECM會促使Drp1蛋白轉至到粒線體，且不利粒線體進行動態變化。ECM所造成的這些現象，並不影響與粒線體動態平衡相關蛋白Drp1與Opa1的蛋白表現量。在我們的實驗中也發現，抗糖尿病藥物會減緩ECM所造成的粒線體膨大與聚集現象。然而，ECM究竟是透過哪一機制調控粒線體動態變化仍不得而知。 利用免疫沈澱反應方法，我們找到了Hiw蛋白會與ECM相交互作用。Hiw是一個E3泛素連接酶，此蛋白已被發現與神經生長有關。綜合以上實驗結果，我們得知ECM各結構區域功能、N端結構對於粒線體膨大聚集化有決定性的功能，並找到與ECM可能參與的訊息路徑。這些結果有助於與ECM相關疾病的治療發展，像是老化疾病及糖尿病。

Drosophila ECM is a CDGSH Ion-Sulfur binding domain containing protein that contains a CDGSH in C-terminal region. In this study, ECM is located on mitochondria at same direction. Overexpression of ECM dramatically causes Enlarged and Clustering Mitochondria in Drosophila S2 cell and mammalian HeLa cell so that we named it as ECM. We defined four regions in ECM, ECM-MH-1 and ECM-MH-2 from their similar and different region as N-terminal, transmembrane region, hinge region and CDGSH region. From the domain swap among ECM-MH-1, ECM-MH-2 and ECM, we found N-terminal of ECM is crucial for the mitochondrial enlargement and clustering in Drosophila and mammalian cell. ECM and ECM-MH-1 are located on mitochondria; the ECM-MH-2 is mainly located on ER in HeLa cell. We exchanged its transmembrane of ECM-MH-2 as the counter domain of ECM-MH-1 and ECM, this fusion ECM-MH-2 protein led to target to mitochondria and also trigger mitochondrial phenotype as same from ECM. We propose that N terminal domain of ECM-MH-2 and its Drosophila ortholog ECM has a critical function in the regulation of mitochondrial enlargement and clustering. In the live imaging, ECM promotes mitochondria to aggregate and cluster then cell divided in the end; it suggest this mitochondrial dynamics phenomenon might be regulated by cell cycle. Overexpression of ECM induced Drp1 to trans-locate on mitochondria and made mitochondria dull to dynamic. But this phenotype did not involve protein expression level of opa1 and drp1. We also found that anti-diabetes drug could reduce mitochondrial enlargement and clustering caused by ECM. However, which mechanism ECM is involved is not clear. By ECM-GFP fusion protein pull-down experiment, we found a protein, Hiw, is interacted with ECM by immunoprecipitation and LC/MS. Hiw, which is an ubiquitin E3 ligase, has been reported to be in the regulation of neuron growth. In this thesis, we evaluate which protein region of ECM that regulates the mitochondrial morphology change. With this clear understanding of ECM structure, function and genetic pathway, it might be possible to develop more therapeutic agents in ECM relative diseases, including aging and diabetes.

INTRODUCTION 1
Iron sulfur protein in organism 1
Iron-sulfur domain-containing proteins, Cisd1, Cisd2 and Cisd3 in mammal 1
CG1458: ortholog protein of Cisd1 and Cisd2 in Drosophila 3
The character of mitochondria in organism 4
Mitochondrial morphology and dynamics 4
MITOCHONDRIA AND DISEASE 6
The aims of the thesis 7
MATERIALS AND METHODS 8
Drosophila S2 cell culture 8
Mammalian HeLa cell culture 8
Transfection 9
Immunofluorescence staining 10
Live cell image 11
Purification of mitochondria 11
Western Blot 12
Drosophila genetics and strains 13
Generation of transgenic flies 13
Drosophila muscle dissection and immunofluorescence 14
RESULTS 15
Define four ECM protein regions 15
ECM is a mitochondria-associated protein and regulates the mitochondrial dynamics 15
Overexpression of mCisd1 and of mCisd2 genes have different mitochondrial morphology phenotypes 16
Hinge region of ECM is dispensable for mitochondrial clustering 17
The long form N-terminal region plays an important role in mitochondrial dynamics 18
The transmembrane region of ECM and mCisd1 might contain a novel mitochondrial targeting sequence (MTS) 20
C-terminal CDGSH region of ECM is dispensable for its mitochondrial enlargement and clusters 21
Time lapse dynamic show ECM induced mitochondrial phenotype might be cell cycle dependent 22
The enlarged and clustering mitochondria is dull for dynamic change, and the protein level of Drp1 and Opa1 remain unchanged 22
Anti-diabetic drug Pioglitazone was dose-dependent on partially suppressing mitochondrial fragmentation induced by ECM in S2 cell and HeLa cell 24
Protein HIW might interact with ECM 25
DISCUSSIONS AND CONCLUSIONS 26
REFERENCE 32
FIGURES 37
TABLES 71

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