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研究生：	林孜謙 Lin, Tzu-Chien
論文名稱：	Septin7做為一種中心體蛋白,藉以調控細胞骨架和生長 Septin 7 as a centrosomal protein that regulates microtubule nucleation and proper cell growth
指導教授：	王家義 Wang, Chia-Yih
學位類別：	碩士 Master
系所名稱：	醫學院 - 細胞生物與解剖學研究所 Institute of Cell Biology and Anatomy
論文出版年：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	61
中文關鍵詞：	septin7 、中心體、中心體蛋白、細胞骨架微管架構、細胞遷移、p150glued
外文關鍵詞：	Septin7, centrosome, centrosomal protein, microtubule nucleation, migration, p150glued
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目前已知在哺乳類動物細胞中，不同亞群的Septin會依照特定的順序彼此鍵結而形成核心複合體，再依此為單位，繼續架構成更高層次的絲狀或找環狀結構。而在這些不同的Septin亞群之中，發現特別Septin7對於影響胚胎的發育以及細胞的分化非常重要，並且Septin7也參與了協調細胞分裂以及細胞遷移的調控過程。在先前的研究當中，也發現Septin7會出現在細胞移動方向的前緣、由細胞骨架actin所構成的stress fiber周圍，因而影響細胞的移動能力，然而Septin7對於細胞生長及遷移之間，其中確切的機制卻仍然不清楚。在本篇研究當中，我們發現Septin7不僅會出現在stress fiber的位置，甚至會位在細胞的中心體上面；並且除了septin7以外，其他的亞群如septin2或septin6都不會出現在中心體上，因此我們推論，septin7是一種中心體蛋白，因而成為影響正常細胞功能的關鍵。我們進而利用多種偵測中心體蛋白的實驗，證實septin7的確是一種中心體蛋白。已知中心體在細胞當中，是維持正常細胞週期的重要角色，也是細胞骨架(微管)的架構中心，因此我們也進一步分析septin7這個中心體蛋白對於細胞功能的影響。從實驗中發現，當細胞週期處在不同進程的時候，中心體上的setpin7表現量也會有明顯的差異。此外，當septin7的表現量被siRNA或者FCF等藥物抑制之後，就會使得中心體結構不穩定，以至於細胞週期無法正常進行，導致細胞生長的數量減少；並且這些septin7表現量低的細胞，其微管的架構能力也會明顯的受到抑制，因而細胞骨架不能正常的幫助中心體在細胞內移動，也會讓高基氏體的結構異常，而大幅的降低了細胞遷移的能力。另外，從實驗室先前的研究中，已經發現另一個也一樣會影響微管架構與細胞週期的中心體蛋白p150glued，因此我們懷疑，septin7可能藉由p150glued去影響細胞功能。從實驗中發現，septin7會和p150形成蛋白複合體，而septin7會透過調控p150蛋白穩定性的方式來影響p150的表現量；並且在抑制p150的表現量之後，會產生與septin7表現量降低時相同的情形，如抑制細胞生長、細胞遷移的能力降低，以及微管架構的異常情形。而當我們在septin7被抑制的細胞中，再送進不同的septin7 construct以及p150glued時，都可以恢復細胞骨架的微管架構能力。此外，從斑馬魚胚胎發育的過程中，也可以明顯的觀察出抑制septin7所導致的發育異常。因此在本篇實驗中，我們認為septin7作為一種中心體蛋白，會透過調控p150glued，而影響正常的細胞生長以及細胞遷移的能力。

By interacting with different Septin subgroup, the mammalian septin core complex is arranged in the order of Septin-7-6-2-2-6-7 hetero-hexametric filament followed by orchestrating high-ordered filaments. Among these Septins, Septin7 is the essential unit of the core complex due to its irreplaceability. In addition, Septin7 is essential for development and differentiation, and coordinates cell division and migration. However, the role of Septin7 in regulating microtubule nucleation is as yet unknown. Here we showed that, in addition to co-localizing with stress fibers, Septin7, but not Septin6 and 2, also localized to the centrosome, the microtubule nucleating center, in a cell cycle-dependent manner. The level of centrosomal septin7 was higher during interphase but gradually reduced at M phase. Depletion of septin7 by siRNA or treated cells with Septin inhibitor forchlorfenuron (FCF), the microtubule nucleation activity was reduced, leading to centrosome mis-orientation and disorganized Golgi ribbon, thus resulting in poor cell migration. In addition, depletion of septin7 inhibited cell proliferation by reducing cyclin E expressions and S phase entry. We then found that the level of p150glued, other centrosomal protein regulates centrosome orientation and Golgi organization, was interacted with Septin7 and downregulated by Septin7. Depletion of p150glued resulted in a phenotype reminiscent of Septin7 deficient cells. To further confirm, we transfected different constructs to rescue the phenotype of nucleation defect caused by knockdown septin7.we found that the construct of septin7, septtin7-GBD and even p150glued can rescued the microtubule structure. Moreover, FCF treatment led to reducing microtubule nucleation and cell migration in zebrafish embryo supporting that Septin7 is essential for embryo development in vivo. Thus, we have uncovered the molecular mechanism by which Septin7 regulates cell growth and migration, at least in part, by controlling microtubule nucleation via p150glued.

目錄
論文考試合格證明書	I
中文摘要	II
ABSTRACT	IV
ACKNOWLEDGEMENT	VI

INTRODUCTION	1
1.The functions of Septins	1
2.The functions of centrosome	2
3.Microtubule nucleation	3
4.The functions of P150glued	4

MATERIAL and METHOD	6

RESULTS	11
A.Septin7 is a centrosomal protein.	11
B.Sepin7 does not affect the structure of centrosome.	12
C.The expression pattern of centrosomal septin7 is cell cycle dependent.	12
D.Septin7 regulates proper cell growth by mediating S phase entry	13
E.Septin7 is important for centrosome homeostasis	14

DISCUSSION	19

References	22

Figures	25
Figure 1. Septin7 localized on the basal body of primary cilia and co-localized with the centrosome	25
Figure 2. Only septin7 but other Septins is co-localized on the centrosome	26
Figure 3. Exogenous Septin7 can also localized on the centrosome	27
Figure 4. The expression level of Septin7 on the centrosome could be knockdown by specific siRNA	28
Figure 5. The signal of centrosomal Septin7 is independent on the microtubule transportation	29
Figure 6. Septin7 could be detected in the same fraction of centrosome extraction by the centrosome fractionation	30
Figure 7. Septin7 depletion did not destroy the structure of centrosome	31
Figure 8. The expression of septin7 variated at different cell cycle stage	32
Figure 9. The expression of septin7 variated at different cell cycle stage during M phase	33
Figure 10. The total protein level of septin7 in the cell was remained stable at different cell cycle stage	34
Figure 11. Septin7 depletion inhibit the cell growth	35
Figure 12. Septin7 depletion reduces the ability of S phase entry	36
Figure 13. Septin7 depletion reduces the ability of S phase entry	37
Figure 14. The ability of S phase defect could be rescued by introducing Septin7 constructs.	38
Figure 15. Septin7 mediates centrosome homeostasis	40
Figure 16. Septin7 depletion caused centrosome amplification is CDK dependent	41
Figure 17. Multiple centrosome caused by Septin7 led to irregular mitotic spindle pore	42
Figure 18. Septin7 is required for microtubule nucleation	43
Figure 19. Septin7 depletion inhibits cell migration	44
Figure 20. Septin inhibitor blocks the ability of cell migration	45
Figure 21. Septin7 depletion inhibits both the distance and orientation of cell migration	46
Figure 22. Septin7 depletion increases the ratio of mis-leading centrosome in migrating cell	47
Figure 23. Septin7 depletion leads to disrupted Golgi ribbon organization	48
Figure 24. The expression level of p150 is inhibited in septin7 depletion cells	49
Figure 25. The expression level of Septin7 in septin7 depletion cells	50
Figure 26. The protein-protein interaction between Septin7 and p150glued	51
Figure 27. The co-localization of Septin7 and p150glued on centrosome	52
Figure 28. The protein stability of p150 is reduced in Septin7 depletion cells.	53
Figure 29. p150 depletion inhibits the cell growth	54
Figure 30. p150glued depletion also inhibits cell migration	55
Figure 31. p150 is required for microtubule nucleation as well.	56
Figure 32. Nucleation defect could be rescued by Septin7 and P150 construct	58
Figure 33. Septin7 inhibitor FCF reduced cell division in zebrafish embryo.	59
Figure 34. Septin7 inhibitor FCF blocked the ability of embryonic migration.	60
Figure 35. The summary model	61


                                    

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校內：2022-06-30公開
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