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.

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