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研究生:紀厚邑
研究生(外文):Ho-Yi Chi
論文名稱:mCostars蛋白對於細胞爬行及肌動蛋白調控之特性分析
論文名稱(外文):The role of mCostars in cell migration and actin regulation
指導教授:陳美瑜陳美瑜引用關係
指導教授(外文):Mei-Yu Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:42
中文關鍵詞:mCostars蛋白細胞爬行
外文關鍵詞:mCostarscell migration
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中文摘要

mCostars (mammalian Costars)為Costars於哺乳類生物中的同源蛋白。Costars為本實驗室先前於Dictyostelium discoideum中發現、與細胞爬行有關的新穎蛋白;研究結果顯示缺乏Costars的黏菌細胞爬行能力減低,且發現Costars會與F肌動蛋白交互作用、並影響黏菌細胞肌動蛋白的分布及其動態平衡。本論文欲更進一步研究人類mCostars蛋白的功能,探討其對於細胞爬行能力、以及細胞內肌動蛋白之動態平衡的影響。在細胞爬行能力方面,分析結果顯示,不論是利用shRNA使Hs.mCostars表現量下降,或是利用CRISPR技術剔除Hs.mCostars基因,皆可使細胞爬行能力下降;若在這些細胞中重新表現mCostars蛋白,則細胞爬行能力可再被回昇。在Hs.mCostars是否調控肌動蛋白方面,首先對於與肌動蛋白有關的細胞現象進行分析,結果發現不論是Hs.mCostars表現量下降或是剔除Hs.mCostars基因之細胞,其細胞面積皆有變大之現象;若利用Hs.mCostars原始表現量較少之細胞,使其大量表現Hs.mCostars,結果發現平均細胞面積縮小,且細胞型態呈現較圓之現象。隨後進行Hs.mCostars影響細胞內G與F肌動蛋白動態平衡之分析,結果發現當剔除Hs.mCostars時,細胞內F/G肌動蛋白比例下降;若在這些細胞中重新表現mCostars蛋白,則F/G肌動蛋白比例可再被回昇。最後為了研究Hs.mCostars於細胞不同位置之功能,我利用質體使Hs.mCostars僅表現於細胞質或細胞核中,並分析不同位置Hs.mCostars表現對於細胞爬行功能之影響,初步結果發現,穩定表現mCostars蛋白於細胞質中會影響細胞爬行能力,而當表現於細胞核中會影響肌動蛋白之動態平衡;mCostars蛋白如何影響細胞爬行以及肌動蛋白動態平衡之機制尚待釐清,而mCostars蛋白在細胞中不同位置的功能也需要未來繼續研究。

Abstarct

mCostars (mammalian Costars) is the mammalian homologue of Costars, a novel cell migration-related protein previously discovered in Dictyostelium discoideum in our laboratory. Previous results desmonstrate that cell migration is decreased in Dictyostelium cells lacking Costars; evidence also indicates that Costars can interact with F-actin, affect actin organization in Dictyostelium cells, and modulate actin dynamics. In this study, I have further investigated the function of human mCostars, focusing on the role of mCostars in regulating cell migration and cellular actin dynamics. Concerning the role of mCostars in cell migration, our results show that cell migration is decreased in either shRNA-mediated Hs.mCostars expression knockdown cells or in CRISPR-mediated Hs.mCostars knockout cells; over-expressing GFP-mCostars in these cells can increase the cell migration ability. Concerning the role of mCostars in regulating the actin cytoskeleton, I have first analyzed cell morphology, which is an actin-related phenotype, and found that the average cell area is increased in either Hs.mCostars-knockdown or Hs.mCostars-knockout cells; on the other hand, overexpression of GFP-mCostars in cells with low endogenous mCostars levels leads to a increased average cell area and a more round-up cell shape. Furthermore, to understand whether mCostars regulates actin dynamics, I have separated cellular G- and F-actin by ultracentrifuge and found that the cellular F/G actin ratio is decreased in Hs.mCostars-knockout cell; overexpressing GFP-mCostars in these cells results in an increased F/G actin ratio. Finally, I have explored the function of mCostars at different subcellular localization by overexpressing GFP-mCostars in cytosol or the nucleus. Preliminary results have showed that stably expressing cytosolic but not nuclear GFP-mCostars can rescue cell migration. However, stably expressing nuclear but not cytosolic GFP-mCostars can rescue cellular F/G-actin ratio in Hs.mCostars-knockout cells. Further investigation is necessary to elucidate the functions of mCostars in different cellular compartments.

目錄
中文摘要. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .i
Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ii
目錄. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv
壹、 緒論. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
一、細胞爬行(cell migration) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
二、細胞爬行之機轉. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
三、細胞爬行與肌動蛋白之動態平衡. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
四、Dd. Costars與Hs. mCostars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
五、Costars與細胞爬行. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
六、研究目標. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
貳、實驗材料與方法. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
一、實驗材料. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
二、實驗方法. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
參、實驗結果. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
一、Hs.mCostars與癌細胞爬行能力. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
二、Hs.mCostars與肌動蛋白調控之細胞功能. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
三、Hs.mCostars與肌動蛋白之動態平衡. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
四、細胞質與細胞核中Hs.mCostars功能之探討. . . . . . . . . . . . . . . . . . . . . . . . . . .20
肆、討論. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
一、Hs.mCostars與癌細胞之細胞爬行能力. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
二、Hs.mCostars與肌動蛋白調控之現象. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
三、Hs.mCostars與肌動蛋白之動態平衡. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
四、細胞質與細胞核中Hs.mCostars功能之探討. . . . . . . . . . . . . . . . . . . . . . . . . . .23
伍、參考文獻. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
陸、圖表. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30

1.Gillitzer, R., and Goebeler, M. (2001). Chemokines in cutaneous wound healing. J Leukoc Biol 69, 513-521.
2. Koizumi, K., Hojo, S., Akashi, T., Yasumoto, K., and Saiki, I. (2007). Chemokine receptors in cancer metastasis and cancer cell-derived chemokines in host immune response. Cancer Sci 98, 1652-1658.
3. Lecaudey, V., and Gilmour, D. (2006). Organizing moving groups during morphogenesis. Curr Opin Cell Biol 18, 102-107.
4. Moser, B., Wolf, M., Walz, A., and Loetscher, P. (2004). Chemokines: multiple levels of leukocyte migration control. Trends Immunol 25, 75-84.
5. Sheetz, M.P., Felsenfeld, D.P., and Galbraith, C.G. (1998). Cell migration: regulation of force on extracellular-matrix-integrin complexes. Trends Cell Biol 8, 51-54
6. Schmidt, A., and Hall, A. (2002). Guanine nucleotide exchange factors for Rho
GTPases: turning on the switch. Genes Dev 16, 1587-1609.
7. Nobes, C.D., and Hall, A. (1995). Rho, rac, and cdc42 GTPases regulate the assembly
of multimolecular focal complexes associated with actin stress fibers, lamellipodia,
and filopodia. Cell 81, 53-62.
8. Rohatgi, R., Ho, H.Y., and Kirschner, M.W. (2000). Mechanism of N-WASP activation
by CDC42 and phosphatidylinositol 4, 5-bisphosphate. J Cell Biol 150, 1299-1310.
9. Eden, S., Rohatgi, R., Podtelejnikov, A.V., Mann, M., and Kirschner, M.W. (2002).
Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck.
Nature 418, 790-793.
10. Weiner, O.D., Neilsen, P.O., Prestwich, G.D., Kirschner, M.W., Cantley, L.C., and Bourne, H.R. (2002). A PtdInsP(3)- and Rho GTPase-mediated positive feedback loop
regulates neutrophil polarity. Nat Cell Biol 4, 509-513.
11. Ridley, A.J. (2001). Rho proteins, PI 3-kinases, and monocyte/macrophage motility.
FEBS Lett 498, 168-171.
12. Ridley, A.J., and Hall, A. (1992). The small GTP-binding protein rho regulates the
assembly of focal adhesions and actin stress fibers in response to growth factors.
Cell 70, 389-399.
13. Maekawa, M., Ishizaki, T., Boku, S., Watanabe, N., Fujita, A., Iwamatsu, A., Obinata,
T., Ohashi, K., Mizuno, K., and Narumiya, S. (1999). Signaling from Rho to the actin
cytoskeleton through protein kinases ROCK and LIM-kinase. Science 285, 895-898.
14. Kawano, Y., Fukata, Y., Oshiro, N., Amano, M., Nakamura, T., Ito, M., Matsumura, F.,
Inagaki, M., and Kaibuchi, K. (1999). Phosphorylation of myosin-binding subunit
(MBS) of myosin phosphatase by Rho-kinase in vivo. J Cell Biol 147, 1023-1038.
15. Snapper, S.B., Takeshima, F., Anton, I., Liu, C.H., Thomas, S.M., Nguyen, D., Dudley, D., Fraser, H., Purich, D., Lopez-Ilasaca, M., et al. (2001). N-WASP deficiency
reveals distinct pathways for cell surface projections and microbial actin-based
motility. Nat Cell Biol 3, 897-904.
16. Welch, M.D., and Mullins, R.D. (2002). Cellular control of actin nucleation. Annu Rev Cell Dev Biol 18, 247-288.
17. Vinson, V.K., De La Cruz, E.M., Higgs, H.N., and Pollard, T.D. (1998). Interactions of Acanthamoeba profilin with actin and nucleotides bound to actin.
Biochemistry 37, 10871-10880.
18. Kaiser, D.A., Vinson, V.K., Murphy, D.B., and Pollard, T.D. (1999). Profilin is predominantly associated with monomeric actin in Acanthamoeba. J Cell Sci 112
( Pt 21), 3779-3790.
19. Pollard, T.D., Blanchoin, L., and Mullins, R.D. (2000). Molecular mechanisms
controlling actin filament dynamics in nonmuscle cells. Annu Rev Biophys Biomol
Struct 29, 545-576.
20. Kuspa, A., and Loomis, W.F. (1992). Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. Proc Natl Acad Sci U S
A 89, 8803-8807.
21. Pang, T.L., Chen, F.C., Weng, Y.L., Liao, H.C., Yi, Y.H., Ho, C.L., Lin, C.H., and Chen, M.Y. (2010). Costars, a Dictyostelium protein similar to the C-terminal domain of STARS,
regulates the actin cytoskeleton and motility. J Cell Sci 123, 3745-3755.
22. Cramer, L., and Mitchison, T.J. (1993). Moving and stationary actin filaments are
involved in spreading of postmitotic PtK2 cells. J Cell Biol 122, 833-843.
23. Beningo, K.A., Dembo, M., Kaverina, I., Small, J.V., and Wang, Y.L. (2001). Nascent
focal adhesions are responsible for the generation of strong propulsive forces in
migrating fibroblasts. J Cell Biol 153, 881-888.
24. DuFort, C.C., Paszek, M.J., and Weaver, V.M. (2011). Balancing forces: architectural
control of mechanotransduction. Nat Rev Mol Cell Biol 12, 308-319.
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