腔腸動物與渦鞭毛藻的胞內共生(cnidarian-dinoflagellate endosymbiosis)關係，在珊瑚礁生態中扮演著關鍵的角色，不論是在基礎生產力或生物多樣性上都貢獻許多。本論文是研究美麗海葵Rab37蛋白(ApRab37)與油滴的關聯及其在共生關係的角色。Rab 家族蛋白是小分子GTP結合蛋白，主要功能是調控囊泡運輸。ApRab37蛋白質由215個胺基酸組成，與人類Rab37在胺基酸序列上有73.1% 的相同。免疫螢光染色實驗統計約有70.6 % 共生小體族群上有ApRab37蛋白，在哺乳類細胞 (COS7 cells) 的螢光融合蛋白實驗中顯示，ApRab37在細胞核的一側有集中的現象，且在油滴有明顯分佈。細胞免疫螢光染色分析亦顯示，在共生狀態下的美麗海葵，其細胞內的ApRab37與油滴有相同或類似的重疊，另外在ER-ID染色發現ApRab37與內質網亦有類似的情形，因此ApRab37應是內質網與油滴相關之胞器蛋白。藉由nocodazole 破壞微小管發現，綠色螢光蛋白集中在核的一側的情形消失，也觀察不到大型的油滴，因此推測ApRab37的功能有賴於完整的微小管。在BFA處理影響高基氏體的實驗發現，BFA並沒有影響ApRab37集中於核一側，但改變ApRab37與Nile red兩者螢光強度呼應的關係，所以ApRab37與高基氏體應具有相關性。在DCMU實驗顯示，DCMU的處理會影響ApRab37在共生藻的分佈，酵母菌餵食實驗則指出ApRab37在晚期胞噬小體有大部份的分佈。總結，ApRab37可能是油滴相關蛋白，在胞內共生的關係中，將光合作用產生的油滴運送至宿主細胞的機制上，ApRab37可能扮演著重要的角色。

Coral reefs ecosystems are some of the most productive and diverse in the world. The symbiotic association between cnidarians and their symbiotic microalgae is of great importance in coral reef ecosystems; however, its underlying molecular mechanism remains unclear even after decades of research. Rab small GTP binding proteins are critical regulators of vesicle trafficking. Here we present the experimental evidence supporting a possible association of ApRab37 with the surface of lipid droplets in the endosymbiosis between the sea anemone, Aiptasia pulchella and the symbiotic dinoflagellate (commonly known as zooxanthellae). ApRab37, a protein of 215 amino acids, displays strong homology with human Rab37. In transfected COS7 cells, EGFP-ApRab37 localized to lipid droplets and clustered in the peri-nuclear region, which stained positive for the ER (endoplasmic reticulum) marker. Immunostaining analysis found ApRab37 associated with symbiosomes and lipid droplets, which was also confirmed by Western blot analysis of in the enrich lipid droplet fraction. Phagocytosis assay showed that ApRab37 involved in late phase of phagocytosis. DCMU treatment indicates symbiosome association of ApRab37 is mediated by zooxanthellae. I propose that ApRab37 plays a pivotal role in the regulation of lipid trafficking from the symbiosomes to the host cell during the Aiptasia-zooxanthellal endosymbiosis.

謝辭 i
中文摘要 ii
英文摘要 iii
目錄 iv
圖目錄vi
附錄目錄viii
一、前言1
(一) 珊瑚礁生態系及其胞內共生現象1
(二) 腔腸動物與共生藻之胞共生現象於細胞生物學上的特殊意義 2
(三) 共生關係的建立是涉及光合作用產物的輸送3
(四) 胞內共生關係的適應 4
(五) Rab small GTP-binding蛋白5
(六) Lipid Droplets7
(七) 以美麗海葵當作胞內共生研究模式動物8
(八) 研究想法與動機8
二、材料方法9
2.1實驗材料9
2.2研究設計10
2.2實驗方法10
(一) 表現ApRab37重組蛋白(Expression of recombinant ApRab37 proteins ) 10
(二) ApRab37重組蛋白純化12
(三) 硫酸十二酯鈉聚丙烯醯胺膠體電泳（SDS-PAGE）13
(四) COS-7細胞培養與繼代 14
(五) 綠色螢光蛋白(EGFP)功能性分析 14
(六) 西方點墨法(Western blot) 15
(七) 免疫螢光分析16
(八) 酵母菌餵食與DCMU處理 17
(九) 油滴純化分析18
(十) 藥劑處理分析18
三、結果21
(一) ApRab37胺基酸序列分析21
(二) pEGFP- ApRab37細胞轉染分析21
(三) BFA與Nocodazole處理22
(四) ER-ID及PDI染色23
(五) 西方點墨法(Western blot) 23
(六) 免疫螢光分析 24
(七) 酵母菌餵食與DCMU處理24
(八) 油滴之免疫螢光染色分析25
四、討論26
一、 ApRab37胺基酸序列分析26
二、EGFP-ApRab37 功能性分析26
三、BFA 及Nocodazole 處理28
四、ApRab37位於晚期吞噬小體 29
五、美麗海葵免疫螢光分析29
六、ApRab37調控共生小體脂肪輸送到宿主細胞之假說31
七、ApRab37在細胞內的角色31
五、參考文獻 62
附錄 70
圖一、美麗海葵(Aiptasia pulchella) 33
圖二、ApRab37-Q82L及ApRab37-T36N與野生型ApRab37 (ApRab37-WT)
比對分析 34
圖三、美麗海葵中選殖出ApRab37 35
圖四、ApRab37的cDNA序列及預測之胺基酸序列 36
圖五、ApRab37及其他Rab37家族蛋白之胺基酸多重序列比對 37
圖六、ApRab37樹狀親源關係圖 39
圖七、EGFP- ApRab37細胞內分佈分析及Nile red 油滴染色 40
圖八、pEGFP- ApRab37轉染及Nile red染色分析 41
圖九、添加油酸來促進油滴的形成 42
圖十、ApRab37Q82L的細胞內分佈分析 43
圖十一、ApRab37T36N細胞內分佈分析 44
圖十二、pEGFP-ApRab37轉染後之哺乳動物細胞處理BFA 46
圖十三、pEGFP-ApRab37轉染後之哺乳動物細胞以nocodazole處理 48
圖十四、美麗海葵內胚層細胞之內質網分佈分析 49
圖十五、ER-ID染色於pEGFP-ApRab37轉染後之哺乳動物細胞 50
圖十六、pEGFP-ApRab37轉染後之哺乳動物細胞進行PDI免疫染色 51
圖十七、純化之美麗海葵ApRab37重組蛋白 52
圖十八、ApRab37西方點墨分析 53
圖十九、美麗海葵消化細胞之ApRab37免疫螢光染色 54
圖二十、美麗海葵細胞之油滴免疫螢光染色分析 56
圖二十一、ApRab37免疫螢光染色美麗海葵胞噬作用 57
圖二十二、酵母菌餵食美麗海葵之胞噬作用分析 58
圖二十三、DCMU處理美麗海葵59
圖二十四、美麗海葵油滴純化分析 60
圖二十五、ApRab37調控共生小體脂肪輸送到宿主細胞之假說 61
附錄一 70
附錄二: pET-100/D-TOPO載體示意圖 72
附錄三: pEGFP-C1載體示意圖 73
附錄四:研究設計流程 74
附錄五:藥劑處理示意圖 75

Alberts, B. 2002. Molecular biology of The Cell. Garland Science. New York.
Anant, J. S., and coauthors. 1998. Mechanism of Rab geranylgeranylation: formation of the catalytic ternary complex. Biochemistry 37(36):12559-68.
Bartz, R., and coauthors. 2007a. Lipidomics reveals that adiposomes store ether lipids and mediate phospholipid traffic. Journal of Lipid Research 48(4):837-47.
Bartz, R., and coauthors. 2007b. Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation. Journal of Proteomic Research 6(8):3256-65.
Beller, M., and coauthors. 2008. COPI complex is a regulator of lipid homeostasis. PLoS Biology 6(11):e292.
Bertucci, A., E. Tambutte, S. Tambutte, D. Allemand, and D. Zoccola. 2010. Symbiosis-dependent gene expression in coral-dinoflagellate association: cloning and characterization of a P-type H+-ATPase gene. Proceedings Biological sciences 277(1678):87-95.
Biel, K. Y., R. D. Gates, and L. Muscatine. 2007. Effects of free amino acids on the photosynthetic carbon metabolism of symbiotic dinoflagellates. Russian Journal of Plant Physiology 54(2):171-183.
Birkeland, C. 1997. Life and Death of Coral Reefs, Chapman and Hall, New York.
Bostrom, P., and coauthors. 2007. SNARE proteins mediate fusion between cytosolic lipid droplets and are implicated in insulin sensitivity. Nature cell biology 9(11):1286-1293.
Bostrom, P., and coauthors. 2005. Cytosolic lipid droplets increase in size by microtubule-dependent complex formation. Arteriosclerosis, thrombosis, and vascular biology 25(9):1945-51.
Brunner, Y., and coauthors. 2007. Proteomics analysis of insulin secretory granules. Molecular and cellular proteomics 6(6):1007-17.
Chavrier, P., and B. Goud. 1999. The role of ARF and Rab GTPases in membrane transport. Current opinion in cell biology 11(4):466-75.
Chen, C. S., and coauthors. 2010. Proteomic analysis of symbiosome membranes in Cnidaria-dinoflagellate endosymbiosis. Proteomics 10(5):1002-1016.
Chen, C. S., and coauthors. 2009. Ratiometric imaging of gastrodermal lipid bodies in coral–dinoflagellate endosymbiosis. Coral Reefs 28(1):289-301.
Chen, M. C., Y. M. Cheng, M. C. Hong, and L. S. Fang. 2004. Molecular cloning of Rab5 (ApRab5) in Aiptasia pulchella and its retention in phagosomes harboring live zooxanthellae. Biochemical and biophysical research communications 324(3):1024-33.
Chen, M. C., Y. M. Cheng, P. J. Sung, C. E. Kuo, and L. S. Fang. 2003. Molecular identification of Rab7 (ApRab7) in Aiptasia pulchella and its exclusion from phagosomes harboring zooxanthellae. Biochemical and biophysical research communications 308(3):586-95.
Chen, X., S. A. Ernst, and J. A. Williams. 2003. Dominant negative Rab3D mutants reduce GTP-bound endogenous Rab3D in pancreatic acini. Journal of chemical biology 278(50):50053-60.
Cohen, A. L., and T. A. McConnaughey. 2003. Geochemical Perspectives on Coral Mineralization. Reviews in Mineralogy and Geochemistry 54(1):151-187.
Fan, J., C. Andre, and C. Xu. 2011. A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii. FEBS letters.
Falkowski, P. G., Z. Dubinsky, L. Muscatine, and J. W. Porter. 1984. Light and the bioenergetics of a symbiotic coral. Bioscience 34(11):705-709.
Fischer von Mollard, G., and coauthors. 1990. rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles. Proceedings of the National Academy of Sciences of the United States of America 87(5):1988-92.
Fitt, W. K., and R. K. Trench. 1983. Endocytosis of the symbiotic dinoflagellate Symbiodinium microadriaticum Freudenthal by endodermal cells of the scyphistomae of Cassiopeia xamachana and resistance of the algae to host digestion. ournal of cell science 64:195-212.
Furla, P., Z. Dubinsky, N. Stambler, S. Richier, and D. Allemand. 2011. Physiological Adaptation to Symbiosis in Cnidarians. Pages 187-195 in Coral Reefs: An Ecosystem in Transition. Springer Netherlands.
Gagnon, E., and coauthors. 2002. Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages. Cell 110(1):119-31.
Gallwitz, D., C. Donath, and C. Sander. 1983. A yeast gene encoding a protein homologous to the human c-has/bas proto-oncogene product. Nature 306(5944):704-707.
Grant, A. J., M. Rémond, T. Starke-Peterkovic, and R. Hinde. 2006. A cell signal from the coral Plesiastrea versipora reduces starch synthesis in its symbiotic alga, Symbiodinium sp. Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology 144(4):458-463.
Greenberg, A. S., and coauthors. 1991. Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets. Journal of chemical biology 266(17):11341-6.
Hodges, B. D., and C. C. Wu. 2007. Proteomic insights into an expanded cellular role for cytoplasmic lipid droplets. Journal of lipid research 51(2):262-73.
Hohman, T. C., P. L. McNeil, and L. Muscatine. 1982. Phagosome-lysosome fusion inhibited by algal symbionts of Hydra viridis. The Journal of Cell Biology 94(1):56-63.
Holz, R. W., W. H. Brondyk, R. A. Senter, L. Kuizon, and I. G. Macara. 1994. Evidence for the involvement of Rab3A in Ca (2+)-dependent exocytosis from adrenal chromaffin cells. Journal of Biological Chemistry 269(14):10229.
Jackson, C. L. 2009. Mechanisms of transport through the Golgi complex. Journal of cell science 122(Pt 4):443-52.
Jiang, H. P., and G. Serrero. 1992. Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein. Proceedings of the National Academy of Sciences of the United States of America 89(17):7856-60.
Kazandjian, A., and coauthors. 2008. Isolation of symbiosomes and the symbiosome membrane complex from the zoanthid Zoanthus robustus. Journal Information 47(3).
Klausner, R. D., J. G. Donaldson, and J. Lippincott-Schwartz. 1992. Brefeldin A: insights into the control of membrane traffic and organelle structure. The Journal of cell biology 116(5):1071-80.
Klionsky, D. J., and S. D. Emr. 2000. Autophagy as a regulated pathway of cellular degradation. Science 290(5497):1717.
Lee, J. O., and coauthors. 2011. Metformin induces Rab4 through AMPK and modulates GLUT4 translocation in skeletal muscle cells. Journal of cellular physiology 226(4):974-81.
Leung, T., and R. Poulin. 2008. Parasitism, commensalism, and mutualism: exploring the many shades of symbioses. Vie et Milieu 58(2):107-115.
Lewis, J. B. 1977. Processes of organic production on coral reefs. Biological Reviews 52(3):305-347.
Liu, P., and coauthors. 2007. Rab-regulated interaction of early endosomes with lipid droplets. Biochim Biophys Acta 1773(6):784-93.
Marchesan, D., and coauthors. 2003. A phospholipase D-dependent process forms lipid droplets containing caveolin, adipocyte differentiation-related protein, and vimentinina cell-freesystem. Journal of chemical biology 278 (29):27293-300.
Martin, S., K. Driessen, S. J. Nixon, M. Zerial, and R. G. Parton. 2005. Regulated localization of Rab18 to lipid droplets: effects of lipolytic stimulation and inhibition of lipid droplet catabolism. Journal of chemical biology 280(51):42325-35.
Martinez, O., and B. Goud. 1998. Rab proteins. Biochim Biophys Acta 1404(1-2):101-12.
Muscatine, L., and E. Cernichiari. 1969. Assimilation of Photosynthetic Products of Zooxanthellae by a Reef Coral. The Biological bulletin 137(3):506-523.
Muscatine, L., P. G. Falkowski, J. W. Porter, and Z. Dubinsky. 1984. Fate of Photosynthetic Fixed Carbon in Light- and Shade-Adapted Colonies of the Symbiotic Coral Stylophora pistillata. Proceedings of the Royal Society of London Series B-Biological Sciences 222(1227):181-202.
Papina, M., T. Meziane, and R. van Woesik. 2003. Symbiotic zooxanthellae provide the host-coral Montipora digitata with polyunsaturated fatty acids. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 135(3):533-537.
Pfeffer, S. R. 2001. Rab GTPases: specifying and deciphering organelle identity and function. Trends in cell biology 11(12):487-91.
Pol, A., and coauthors. 2004. Dynamic and regulated association of caveolin with lipid bodies: modulation of lipid body motility and function by a dominant negative mutant. Journal of molecular cell biology 15(1):99-110.
Roth LE, Jeon K, Stacey G .1988. Homology in endosymbiotic systems: the term
”symbiosome” In R Palacios, DPS Verma, eds, Molecular Genetics of Plant-Microbe Interactions. Biological Nitrogen Fixation, pp 220-225.
Seckbach, J. 2002. Symbiosis: mechanisms and model systems, volume 4. Kluwer academic publishers.
Segev, N. 2001. Ypt and Rab GTPases: insight into functions through novel interactions. Current opinion in cell biology 13(4):500-11.
Singh, R., and coauthors. 2009. Autophagy regulates lipid metabolism. Nature 458(7242):1131-1135.
Soni, K. G., and coauthors. 2009. Coatomer-dependent protein delivery to lipid droplets. Journal of cell science 122(Pt 11):1834-41.
Souter, D. W., and O. Lindén. 2000. The health and future of coral reef systems. Ocean & Coastal Management 43(8-9):657-688.
Stambler, N., and Z. Dubinsky. 2010. Zooxanthellae: The Yellow Symbionts Inside Animals. Pages 87-106 in Coral Reefs: An Ecosystem in Transition. Springer Netherlands.
Stambler, N., Z. Dubinsky, and J. Seckbach. 2011. Marine Microalgae/Cyanobacteria–Invertebrate Symbiosis. Pages 383-414 in All Flesh Is Grass, volume 16. Springer Netherlands.
Stenmark, H., and V. M. Olkkonen. 2001. The Rab GTPase family. Genome biology 2(5):REVIEWS3007.
Swanson, R., and O. Hoegh-Guldberg. 1998. Amino acid synthesis in the symbiotic sea anemone Aiptasia pulchella. Marine Biology 131(1):83-93.
Swift, L. L., and coauthors. 2005. Microsomal triglyceride transfer protein expression in adipocytes: a new component in fat metabolism. FEBS Lett 579(14):3183-9.
Touchot, N., P. Chardin, and A. Tavitian. 1987. Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library. Proceedings of the National Academy of Sciences of the United States of America 84(23):8210-4.
Trench R. K. 1993. Microalgal–invertebrate symbiosis, a review. Endocytobiosis
and Cell Research 9:135-175.
Trench R. K. 1979. The cell biology of plant–animal symbiosis. Annual Rev Plant
Physiol 30:485–531 Birkeland C (1997) Life and death of coral reefs. Chapman and Hall, New York.
Trench, R. K. 1971. The Physiology and Biochemistry of Zooxanthellae Symbiotic with Marine Coelenterates. III. The Effect of Homogenates of Host Tissues on the Excretion of Photosynthetic Products in vitro by Zooxanthellae from Two Marine Coelenterates. Proceedings of the Royal Society of London Series B-Biological Sciences 177(1047):251-&.
Tsuboi, T., and M. Fukuda. 2006. Rab3A and Rab27A cooperatively regulate the docking step of dense-core vesicle exocytosis in PC12 cells. Journal of cell science 119(Pt 11):2196-203.
Venn, A. A., J. E. Loram, and A. E. Douglas. 2008. Photosynthetic symbioses in animals. Journal of Experimental Botany 59(5):1069-1080.
Wang, J. T., and A. E. Douglas. 1997. Nutrients, signals, and photosynthate release by symbiotic algae - The impact of taurine on the dinoflagellate alga Symbiodinium from the sea anemone Aiptasia pulchella. Plant Physiology 114(2):631-636.
Wetterau, J. R., K. A. Combs, L. R. McLean, S. N. Spinner, and L. P. Aggerbeck. 1991. Protein disulfide isomerase appears necessary to maintain the catalytically active structure of the microsomal triglyceride transfer protein. Biochemistry 30(40):9728-35.
Weis, V. M., S. K. Davy, O. Hoegh-Guldberg, M. Rodriguez-Lanetty, and J. R. Pringle. 2008. Cell biology in model systems as the key to understanding corals. Trends in ecology & evolution 23(7):369-76.
Wolins, N. E., B. Rubin, and D. L. Brasaemle. 2001. TIP47 associates with lipid droplets. Journal of chemical biology 276(7):5101-8.
Yellowlees, D., T. A. V. Rees, and W. Leggat. 2008. Metabolic interactions between algal symbionts and invertebrate hosts. Plant, Cell & Environment 31(5):679-694.
Zerial, M., and H. McBride. 2001. Rab proteins as membrane organizers. Nature reviews. Molecular cell biology 2(2):107-17.