臺灣博碩士論文加值系統

TbPF20是布魯氏錐蟲（Trypanosoma brucei）的鞭毛蛋白同源物。RNAi實驗顯示出TbPF20是布魯氏錐蟲生長以及鞭毛功能的必須基因，tbPF20包含第160到230個胺基酸的 SMC功能區以及位於C端第301到589個胺基酸的WD40重複序列，建構出剔除SMC功能區，但包含WD40重複序列的PF20突變蛋白並接上eGFP標記質體，稱之為pSACⅡ，可以表現在哺乳動物上皮細胞。另一個包含完整的WD40重複序列的質體pWDN。本實驗中，這樣截短化（truncated）的tbPF20可被西方墨點法所偵測，也都對細胞具毒性。將pSACⅡ轉染進MDCK細胞中可以產生兩種螢光蛋白族群，一種會表現出精緻的點狀，細緻的沿著細胞邊緣圍住整顆細胞。另一種表現在整顆細胞。點狀族群之螢光細胞在轉染後24小時便死亡。點狀蛋白與肌動蛋白、偽足和細胞黏著分子並沒有相關性。表現SACⅡ與WDN螢光蛋白的細胞微管卻在細胞邊緣表現出捲曲以及形成束狀，而點狀族群的蛋白表現還與劍蛋白p60共同存在的現象，均質狀族群則沒有。由曠時螢光顯微鏡以及metamorph軟體可以計算出，點狀族群與均質狀族群的移動距離與速率都會下降。不僅如此，SACⅡ螢光蛋白細胞經由有絲分裂阻滯劑nocodazole處理16小時後，DNA濃縮百分比小於1%，且沒有核膜破裂與中心體分離的現象。看來，SACⅡ以及WDN蛋白均會擾亂微管以及細胞週期，使中心體無法分離引起 G2/M 停滯造成細胞死亡。這些蛋白如何透過微管系統造成中心體無法分離的現象，進而影響有絲分裂期，需要進一步的討論與研究。

Gene TbPF20 is a PF20 ortholog from flagellate parasite Trypanosoma brucei. RNAi experiment shows TbPF20 is essential both for flagellum function and for parasite proliferation. It contains both SMC domain from residue 160 to 230 and a WD domain with 7 repeats, which locates entire C terminal half from residue 301 to 589. A PF20 deletion mutant plasmid （pSACⅡ） bearing WD repeats with eGFP tag readily expressed in mammalian cells was constructed by deleting SMC domain. The other PF20 deletion construct was pWDN, which comprises the whole WD repeats only. These truncated TbPF20 mutants were transfected into mammalian MDCK cells separately and the corresponding proteins were detected by Western blotting. Both DNA constructs conferred cytotoxicity in MDCK. Interestingly, two populations of fluorescent protein patterns were identified in MDCK cells transfected with pSAC. One gave delicate puncta, thin thread-like beads along cell edges and the other homogeneous throughout. The puncta were not in line with cortical actin, lamellapodia and cell adhesion molecular. Both pSACII and pWDN transfectant cells reduced their mobility in terms of speed and distance from control cells ,respectively as calculated from time lapse data with metamorph program. Both constructs blocked cell cycle progression at the centrosome disjunction step. Both TbPF20 derivative transfectants gave less than 1% round-up shape after 16hr nocodazole treatment in contrast to more than 90% that of the control cells. All these data pointed cytoskeleton abnormality. Phalloidin, E-cadherin, β-catenin stainings all showed no obvious difference from control cells. However, microtubules were drastically abnormal in over 80% cells with SACII or WDN. Microtubules in these cells showed bundling and curling or obviously dimished as the effect of heavy microtubule severing. Confocal microscopy method showed that indeed the thin-thread beads like puncta from pSACII colocalized with Katanin p60, which gives similar pattern of microtubule system upon RNAi Katanin. The homogenous population of pSACII or pWDN did not give colocalization with Katanin p60. From time lapse data, these puncta cell population gave PI staining 24hr post transfection earlier than that of the homogenous SACII or WDN population. More, SacⅡ containing cells showed uncondensed DNA upon nocodazole treatment, and nuclear envelope were not breakdown to disperse into cytoplasm as judged by lamin A/C staining. On the whole, the TbPF20 derivate proteins disorganized microtubules as well as perturbed cell cycle progression at centrosome disjunction to cause G2/M arrest and, thereafter, cytotoxicity. How these proteins mediate the interaction with microtubule system to the centrosome disjunction and further mitosis events awaits further investigation.

目錄

中文摘要 Ⅱ
英文摘要 Ⅲ
致謝 Ⅳ
目錄 Ⅵ
圖目錄 Ⅸ
附錄 Ⅹ
緒論 1
一、布魯氏錐蟲（Trypanosoma brucei） 1
¬ 二、鞭毛與纖毛（Flagella and Cilia） 1
三、鞭毛相關蛋白PF20（Paralyzed flagella protein 20） 2
四、 WD40重複蛋白（WD40 repeats） 3
五、微管 （microtubule） 3
六、劍蛋白（katanin） 4
七、哺乳類動物細胞之有絲分裂（mitosis） 5
八、中心粒（centriole）與中心體（centrosome）週期 7
九、研究目標 7
材料與方法 9
一、材料 9
（一）細胞 9
（二）外購試劑 9
（三）耗材 11
（四）儀器 12
（五）配置試劑 12
二、方法 14
（一） TbPF20預測功能區螢光質體（SACⅡ與WDN）建構 14
（二） 質體的萃取 15
（三） 細胞培養 15
（四） 西方墨點法 16
（五）定時拍攝試驗（Time course） 18
（六）曠時攝影試驗（Time lapse） 18
（七）細胞同步化（synchronization）評估 18
（八）細胞染色 19
結果 22
一、SACⅡ與WDN蛋白經轉譯後表現於細胞質與細胞核 22
二、SACⅡ與WDN蛋白的表現對細胞具毒殺效應 22
三、SACⅡ與WDN蛋白表現細胞使得細胞移動性明顯下降 24
四、SACⅡ與WDN蛋白造成細胞微管的異常 24
五、SACⅡ與細胞內劍蛋白p60有共同存在的現象 26
六、表現SACⅡ與WDN的螢光細胞經Nocodazole 處理之染色體無濃縮現象 26
七、SACⅡ與WDN螢光細胞經Nocodazole處理後失去束狀、捲曲狀以及被切解
微管 27
八、表現SACⅡ與WDN之螢光細胞經Nocodazole 處理之細胞核膜沒有破裂現象 28
九、表現SACⅡ與WDN之螢光細胞的成對中心體無法有效移動 28
討論 30
一、截短化PF20：SACⅡ與WDN蛋白對細胞造成的結果不盡相同 30
二、SACⅡ與WDN蛋白所表現的毒殺效應 31
三、同樣含有WD40重複序列的蛋白所表現的功能與SACⅡ、WDN之比較 32
四、SACⅡ和WDN蛋白與微管的關係 33
五、SACⅡ與WDN的WD40重複序列影響到中心體分離的可能途徑，以及與疾
病關係 34
六、SACⅡ與WDN蛋白擾亂細胞週期的另一可能：CDK1 35
參考文獻 36

圖目錄

圖1 pSACⅡ、pWDN質體轉殖MDCK細胞後螢光蛋白表現型有兩種族群 43
圖2 pSACⅡ點狀細胞族群數量於轉殖後24小時有急遽下降的趨勢 45
圖3 pSACⅡ轉殖MDCK後的點狀細胞幾乎均於轉殖後24小時內死亡 46
圖4 pSACⅡ點狀細胞的蛋白從細胞周圍往細胞核聚集後引起死亡現象 47
圖5 pSACⅡ與pWDN轉殖後螢光蛋白細胞之移動距離與速率均有下降現象 48
圖6 SACⅡ、WDN引起細胞死亡現象與鈣黏著素E、β-連鎖蛋白並無關聯 50
圖7 SACⅡ、WDN引起細胞死亡現象與F-肌動蛋白並無關聯 51
圖8 表現SACⅡ、WDN的細胞出現微管束狀、捲曲狀或被切割之現象 53
圖9 SACⅡ與微管有部分位置重疊現象 54
圖10 SACⅡ與劍蛋白p60有共同存在的現象 55
圖11 Nocodazole處理MDCK使得細胞停在G2/M期，釋放藥物之後G1及S
期有回升現象 57
圖12 0.4μg/ml Nocodazole為使細胞染色體濃縮之最佳濃度 58
圖13 SAC與WDN蛋白螢光表現細胞的染色體皆無法濃縮 60
圖14 SACⅡ與WDN螢光細胞經Nocodazole處理後失去束狀、捲曲狀以及被
切解微管 61
圖15 SACⅡ與WDN螢光表現細胞經Nocodazole處理後細胞核膜蛋白不散置胞質
62
圖16 SACⅡ與WDN螢光表現細胞之中心體無法分離 63
圖17 同步化後的轉殖細胞內成對中心體間距測量長度比例圖 64


附錄

附錄1 WD重複序列之結構 65
附錄2 CDK1 / cyclin B活化路徑 66
附錄3 MDCK細胞在G2晚期與有絲分裂各亞期的中心體間距平均值 67
附錄4 有絲分裂期前中心體分離作用之酵素催化途徑 68
附錄5 纖毛或鞭毛缺失之疾病 69

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