The study of SMC deleted tbPF20 in mammalian epithelial cell

• Main Authors: Yung-HanYang, 楊詠涵
• Other Authors: Chih-Li Hsu
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/21035882895989253625

碩士 === 國立成功大學 === 微生物及免疫學研究所 === 100 === 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.