在海洋中腔腸動物（cnidarian）與共生藻（zooxanthellae）所形成的胞內共生現象，是珊瑚礁生態系中十分重要的基礎生產力貢獻者。這樣的共生關係是如何建立？相關的機制又為何？目前已有相關研究顯示在endocytosis過程的主要調控蛋白－Rab5（early endocytic phase regulating protein）和Rab7 (late endocytic phase regulating protein)，可能扮演相當重要的角色。本研究主要是從細胞分子生物學的角度來探討細胞中的Rab proteins－Rab5和Rab7在Aiptasia-Symbiodinium共生現象之關連性。Rab蛋白質在氨基酸序列上具演化保留性，實驗中利用退化反轉錄聚合連鎖反應（degenerate RT-PCR）和cDNA端點反應（RACE）找到Aiptasia Rab5和Rab7（ApRab5和ApRab7）蛋白質序列，其氨基酸序列與人類Rab5C和Rab7十分相近，在以微珠（latex bead）所進行之phagocytosis，顯示ApRab5主要分佈在前30分鐘的phagosomes上；ApRab7要分佈在30-120分鐘的phagosomes上。過去已知光合作用抑制劑－DCMU會破壞共生關係，因此本研究以免疫染色法比較有無DCMU處理，其包著共生藻的phagosomes （又稱為共生小體）上ApRab5和ApRab7之分佈比率。結果發現未經DCMU處理之大部份共生小體上有ApRab5分佈（~65 %），但少有ApRab7之存在（~35 %）；在經DCMU處理後一小時，有ApRab5分佈的共生小體數量銳減（~30 %），而有ApRab7分佈的共生小體數目卻等比例增多（~70 %）。餵食共生藻實驗中顯示－餵食活的共生藻二小時後，共生小體上約有65 %的ApRab5分佈；ApRab7的分佈僅約36 %。餵食死的共生藻，二小時後共生小體上ApRab5分佈降至約30 %；ApRab7的分佈遽升到約70 %。由上述種種實驗數據顯示，我們推測共生藻成功生存在宿主細胞中可能是藉由使Rab5停留在共生小體上及排除Rab7的作用，使共生小體停留在類似early phagosome stage階段。

Marine cnidarian-microalgal endosymbiosis is an ecologically important intracellular association. However, its underlying molecular mechanisms are essentially unknown. In light of the critical roles of host phagocytosis in intracellular fates of a variety of microbes, and the Rab small GTPases as key mediators of host-symbiont interaction, we set out to investigate the potential involvement of Aiptasia Rab proteins in the model photosynthetic endosymbiosis between the sea anemone, Aiptasia pulchella and the symbiotic dinoflagellate (commonly called zooxanthellae), Symbiodinium spp. Many Aiptasia Rab homologue-encoding cDNA fragments were first cloned through our degenerate RT-PCR and RACE reactions. Significantly, Aiptasia homologues of Rab5 and Rab7 (ApRab5 and ApRab7), two Rabs known to be critical regulators of phagosome maturation were also identified in the screen. The overall sequence identities of ApRab5 and ApRab7 to those of human Rab5C and Rab7 were very extensive, and EGFP reporter, protein fractionation, and immuno-fluorescence studies all suggested that the similarity of the Aiptasia Rabs to their human counterparts extended to the functional levels. Finally, although the phagosomes enclosing latex beads stained positive for ApRab5 and ApRab7 with kinetics characteristics of normal phagosomal maturation, the phagosomes housing zooxanthellae only stained positive for ApRab5. Furthermore, the association of ApRab5 with and the exclusion of ApRab7 from the zooxanthellae-containing phagosomes could be reversed by the heat-killed or photosynthesis-impaired symbionts. Overall, our present study has identified ApRab5 and ApRab7 as potential key regulators of the Aiptasia-Symbiodinium endosymbiosis

一、緒論
（一）細胞藉由胞飲作用擷取細胞外物質
（二）Rab蛋白調控細胞中囊泡運輸
（三）Rab5和Rab7調控胞飲作用的囊泡運送過程及胞噬泡成熟過程
（四）細胞中胞內微生物生存機制
（五）胞內共生藻生存在一個類似胞噬泡的胞器中，與溶小體沒有融合現象
（六）Mycobacterium和Salmonella藉由干擾胞噬泡成熟，以達到胞內寄生目的
（七）研究構想與方向
二、材料與方法
（一）細胞與動物
（二）ApRab5和ApRab7 cDNA選殖與序列分析
（三）ApRab5和ApRab7重組蛋白與純化
（四）蛋白質免疫分析
（五）EGFP-ApRab5和EGFP- ApRab7於哺乳類細胞內之定位
（六）胞噬作用
（七）免疫螢光分析
三、結果
（一）ApRab5和ApRab7全長cDNA選殖與分析
（二）ApRab5和ApRab7蛋白質序列分析
（三）ApRab5位於早期endocytic compartments，會促進early endosomes融合; ApRab7位於晚期、酸性endocytic compartments
（四）ApRab5和ApRab7同時存在海葵細胞細胞質（cytosolic form）及膜器（intracellular membrane）中
（五）ApRab5主要分佈在early phagosomes；
ApRab7主要分佈在late phagosomes
（六）不同控制下ApRab5和ApRab7在phagosomes上的分佈
四、討論與結論
（一）ApRab5和ApRab7與脊推動物Rab5和Rab7蛋白質序列具高度相似性
（二）在哺乳類細胞中EGFP-ApRab5分佈在EE；EGFP-ApRab7分佈在LE
（三）美麗海葵細胞質與細胞膜器皆有ApRab5和ApRab7分佈ApRab5和ApRab7參與phagocytic pathway
（四）ApRab5和ApRab7參與phagocytic pathway（五）ApRab5和ApRab7參與共生藻在腔腸生物消化細胞胞內生存機制調控
五、參考文獻

Aerial. 1992. The small GTPase Rab5 functions as a regulatory factor in the early endocytic pathway. Cell 70:715-728.
Amer, A. O., and M. S. Swanson. A phagosome of one’s own: a microbial guide to life in the macrophage. Current Opinion in Microbiology 5:56-61.
Birkeland, C. 1997. Life and death of coral reefs. Chapman and Hall, New York, USA.
Bucci, C., R. G. Parton, I. H. Mather, H. Stunnenberg, K. Simons, B. Hoflack, and M. Zerial. 1992. The small GTPase Rab5 functions as a regulatory factor in the early endocytic pathway. Cell 70:715-728.
Bucci, C., P. Thomsen, P. Nicoziani, J. McCarthy, and B. van Deurs. 2000. Rab7: a key to lysosome biogenesis. Molecular Biology of the Cell 11:467-480.
Brumell, J.H., P. Tang, S.D. Mills, and B.B. Finlay. 2001. Characterization of Salmonella-induced filaments (Sifs) reveals a delayed interaction between Salmonella-containing vacuoles and late endocytic compartments. Traffic 2:643-653.
Bucheier N. A., and F. Heffron. 1991. Inhibition of macrophage phagosomes -lysosome fusion by Salmonella typhimurium. Infection and Immunity 59:2232-2238.
Clemens, D.L., B. Y.Lee, and M. A. Horwitz. 2000. Mycobacterium tuberculosis and Legionella pneumophila phagosomes exhibit arrested maturation despite acquisition of Rab7. Infection and Immunity 68:5154-5166.
Clemens, D.L., B. Y.Lee, and M. A. Horwitz. 2002. The Mycobacterium tuberculosis phagosome in human macrophages is isolated from the host cell cytoplasm. Infection and Immunity 70:5800-5807.
Courret, N., C. Fréhel, N. Gouhier, M. Pouchelet, E. Prina, P. Roux, and J. C. Antoine. 2002. Biogenesis of Leishmania-harbouring parasitophorous vacuoles following phagocytosisof the metacyclic promastigote or amastigote stages of the parasites. Journal of Cell Science 115:2303-2316.
David, C. N. 1973. A quantitative method for maceration of hydra tissue. Roux's Archives of Developmental Biology 171:259-268.
Desjardins M., L. A. Huber, R. G. Parton, and G. Griffiths. 1994. Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus. Journal of Cell Biology 124: 677-688.
Duclos, S., R. Diez, J. Garin, B. Papadopoulou, A. Descoteaux, H. Stenmark, and M. Desjardins. 2000. Rab5 regulates the kiss and run fusion between phagosomes and endosomes and the acquisition of phagosome leishmanicidal properties in RAW 264.7 macrophages. Journal of Cell Science 113:3531-3541.
Feng, Y., B. Press, and A. Wandinger-Ness. 1995. Rab7: an important regulator of late endocytic membrane traffic. Journal of Cell Biology 131:1435-1452.
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 andresistance of the algae to host digestion. Journal of Cell Science 63:195-212.
Frohman, M. A., M. K. Dush, and G. R. Martin. 1988. Rapid production of full-length cDNAs from rate transcripts: amplification using a single gene-specific oligonucleotide primer. Proceedings of the National Academy of Sciences of the United States of America 85:8998-9002.
Gorvel, J. P., P. Chavrier, M. Zerial, and J. Gruenberge. 1991. Rab5 controls early endosome fusion in vitro. Cell 64:915-925.
Hashim, S., K. Mukherjee, M. Raje, S. K. Basu, and A. Mukhopadhyay. 2000. Live Salmonella modulate expression of Rab proteins to persist in a specialized compartment and escape transport to lysosomes. Journal of Biological Chemistry 275:16281-16288.
Heinzen, R. A., M. A. Scidmore, D. D. Rockey, and T. Hackstadt. 1996. Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis. Infection and Immunity 64:796-809.
Hohman, T. C., P. L. McNeil, and L. Muscatine. 1982. Phagosome-lysosome fusion inhibited by algal symbionts of Hydra Viridis. Journal of Cell Biology 94:56-63.
Hofmann, D. K., and B. P. Kremer. 1981. Carbon metabolism and strobilation in Cassipea andromeda (Cnidaria: Scuphzoa): significance of endocytosymbiotic dinoflagellates. Marine Biology 65:25-33.
Jahraus, A., T. E. Tjelle, T. Berg, A. Habermann, B. Storrie, O. Ullrich, and G. Griffiths. 1998. In vitro fusion of phagosomes with different endocytic organelles form J774 macrophages. Journal of Biological Chemistry 263:30379-30390.
Laura, E. V., D. Deretic, R. J. Ulmer, N. S. Hibler, L. A. Huber, and V. Deretic. Arrest of Mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by Rab5 and Rab7. Journal of Biological Chemistry 272:13326-13331.
Marsh, M. 2001. Endocytosis. Oxford University Press, New York, USA.
Mellman, I. 1996. Endocytosis and molecular sorting. Annual Review of Cell and Developmental Biology 12: 575-625.
Méresse, S., O. Steele-Mortimer, B B. Finlay, and J. P. Gorvel. 1999.The rab7 GTPase controls the maturation of Salmonella typhimurium-containing vacuoles in HeLa cells. EMBO Journal 18:4394-4403.
Mukherjee, S., R. N. Gosh, and F. R. Maxfield. 1997. Endocytosis. Physiological Reviews 77:759-802.
Mukherjee, K., S. Parashuraman, G. Krishnamurthy, J. Majumdar, A. Yadav, R. Kumar, S. K. Basu, and A. Mukhopadhyay. 2002. Diverting intracellular trafficking of Salmonella to the lysosome through activation of the late endocytic Rab7 by intracellular delivery of muramyl dipeptide. Journal of Cell Science 115:3693-3701.
Nagai, H., J. C. Kagan, X. Zhu, R. A. Kahn, and C. R. Roy. 2002. A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes. Science 295:679-682.
Novick, P., and M. Zerial. 1997. The diversity of Rab proteins in vesicle transport. Current Opinion in Cell Biology 9:496-504.
Pereira-Leal, J. B., and M. C. Seabra. 2000. The mammalian Rab family of small GTPases: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily. Journal of Molecular Biology 301:1077-1087.
Pereira-Leal, J. B., and M. C. Seabra. 2001. Evolution of the Rab family of small GTP-binding proteins. Journal of Molecular Biology 313:889-901.
Pitt, A., L. S. Mayorga, P. D. Stahl, and A. L. Atchwartz. 1992. Alterations in the protein composition of maturing phagosomes. Journal of Clinical Investigation 90:1978-1983.
Russell, D. G., S. Sturgill-Kozycki, T. Vanheyningen, H. Collins, and U. E. Schaible. 1997. Why intracellular parasitism need not be a degrading experience for Mycobacterium. Philosophical Transactions: Biological Sciences 352:1303-1313.
Roberts, R. L., M. A. Barbieri, K. M. Pryse, M. Chua, J. H. Morisaki, and P. D. Stahl. 1999. Endosome fusion in living cells overexpressiong GFP-Rab5. Journal of Cell Science 112:3667-3675.
Scianimanico, S., M. Desrosiers, J. F. Dermine, S. Méresse, A. Descoteaux, and M. Desjardins. 1999. Imparied recrutment of the the small GTPase Rab7 correlates with the inhibition of phagosomes maturation by Leishmania donovani promastigotes. Cellular Microbiology 1:19-32.
Scott, C.C., P. Cuellar-Mata, T. Matsuo, H. W. Davidson, and S. Grinstein. 2002. Role of 3-phosphoinositides in the maturation of Salmonella-containing vacuoles within host cells. Journal of Cell Biology 277:12770-12776.
Scott, C. C., R. J. Botelho, and S. Grinstein. 2003. Phagosome maturatiom: A few bugs in the system. Journal of Membrane Biology 193:137-152.
Soldati, T., C. Rancaño, H. Geissler, and S. R. Pfeffer. 1995. Rab7 and Rab9 are recruited onto late endosomes by biochemically Distinguishable processes. Journal of Biological Chemistry 270:25541-25548.
Sönnichsen, B., S. D. Renzis, E. Nielsen, J. Rietdorf, and M. Zerial. 2000. Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of rab4, rab5, and rab11. Journal of Cell Biology 149:901-913.
Stein, M. P., J. Dong, A. Wanding-Ness. 2003. Rab proteins and endocytic trafficking: potential targets for therapeutic intervention. Advanced Drug Delivery Reviews 55:1421-1437.
Stenmark, H., and V. M. Olkkonen. 2001. The Rab GTPase family. Genome Biology 2:3007.1-3007.7.
Sturgill-Koszycki, S., and M. S. Swanson. 2000. Legionella pneumophila replication vacuoles mature into acidic, endocytic organelles. Journal of Experimental Medicine 192:1261-1271.
Thompson, J. D., D. G. Higgins and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22:4673-4680.
Via, L.E., D. Deretic, R. J. Ulmer, N. S. Hibler, L. A. Huber, and V. Deretic. 1997. Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7. Journal of Cell Biology 272:13326-13331.
Zerial, M., and H. McBride. 2001. Rab proteins as membrane organizers. Nature Reviews Molecular Cell Biology 2:107-117.