PF20全長cDNA取自於可造成人類非洲昏睡病（African sleeping sickness）以及動物非洲錐蟲病（nagana）的布魯氏錐蟲（Trypanosoma brucei）。PF20為布魯氏錐蟲和萊茵衣藻（Chlamydomonas reinhardtii）軸絲中央微管連結之必須蛋白。NCBI蛋白質比對軟體（Blastp）比對589個胺基酸序列開放讀序架被預測其帶有SMC（Structure Maintenance of Chromosome）和WD40 domain。將兩個domain建構至eGFP質體並表現於真核細胞後，可發現此蛋白表現於細胞質。定時拍攝和曠時攝影數據上顯示此蛋白對於上皮細胞具有細胞毒性且於40小時內無融合蛋白表現型細胞進入有絲分裂中期。西方墨點法也可看出此融合多肽蛋白之大小為90kD且穩定表現eGFP之控制組細胞株螢光蛋白表現量為其二百倍。藉由nocodazole將細胞阻滯於有絲分裂前中期，發現表現融合蛋白的細胞已具有完成複製後的四套體DNA，但免疫化學染色顯示轉殖的實驗組細胞皆無染色體濃縮、組織蛋白H3磷酸化、轉錄因子Sp1退出、核膜瓦解或中心體分離。根據這些實驗結果顯示單細胞布魯氏錐蟲基因在高等真核細胞內具有細胞毒殺效應，且TbPF20的表現阻滯了中心體的分離，使細胞無法脫離G2期進入有絲分裂期。

Full length cDNA of PF20 was obtained from parasite Trypanosoma brucei, causative agent of African sleeping sickness in humans and nagana in animals. It is an essential gene of the parasite and a bridging protein of central tubules of axoneme in Chlamydomonas reinhardtii. NCBI Protein Blast Program identified SMC domain and WD-repeats domain within the ORF of 589 residues. When plasmid construct containing these two domains fused with eGFP and expressed in mammalian cell, the polypeptide localized cytoplasmically. Time-course and time-lapse data showed that the protein conferred cytotoxicity on MDCK. Western blotting showed the fusion polypeptide was 90kD in size and the expression amount was less than two hundredth that of control vector eGFP from stable clone. Synchronization with nocodazole, fusion protein expressed cells did not reach neither chromosome condensation as judged by histone H3 Ser10 phosphorylation and nucleus devoid of SP1, nor nuclear envelope breakdown judged by lamin A/C staining, or centrosome disjunction judged by distance between γ-tubulin stained centrosome pairs. It seems that ectopic expression of parasitic PF20 could perturb cell cycle progression at centrosome disjunction to cause G2/M arrest and, thereby, cytotoxicity. According to these finding, all these data support the notion that proteins of flagellum have functions other than motility.

(1) Absalon, S., Blisnick, T., Bonhivers, M., Kohl, L., Cayet, N., Toutirais, G., Buisson, J., Robinson, D. and Bastin, P. (2008). Flagellum elongation is required for correct structure, orientation and function of the flagellar pocket in Trypanosoma brucei. J Cell Sci. 121, 3704-3716.
(2) Absalon, S., Kohl, L., Branche, C., Blisnick, T., Toutirais, G., Rusconi, F., Cosson, J., Bonhivers, M., Robinson, D. and Bsatin, P. (2007). Basal Body Positiong Is Controlled by Flagellum Formation in Trypanosoma brucei. PLoS One. 5, 1-16.
(3) Ben, Amar. M. F., Pays, A., Tebabi, P., Dero, B., Seebeck, T., Steinert, M. and Pays, E. (1988). Structure and transcription of the actin gene of Trypanosoma brucei. Mol Cell Biol. 8, 2166-2176.
(4) Bettencourt-Dias, M. and Glover, D. M. (2007). Centrosome biogenesis and function: centrosomics brings new understanding. Nat Rev Mol Cell Biol. 8, 451-463.
(5) Branche, C., Kohl, L., Toutirais, G., Buisson, J., Cosson, J. and Bastin, P. (2006). Conserved and specific functions of axoneme components in trypanosome motility. J Cell Sci. 119, 3443-3455.
(6) Chuang, J. Y., Wang, Y. T., Yeh, S. H., Liu, Y. W., Chang, W. C. and Hung, J. J. (2008). Phosphorylation by c-Jun NH2-terminal Kinase 1 Regulates the Stability of Transcription Factor Sp1 during Mitosis. Mol Biol Cell. 19, 1139-1151.
(7) Cooper, S. (2003). Rethinking synchronization of mammalian cells for cell cycle analysis. Cell Mol Life Sci. 60, 1-9.
(8) Cunha-Ferreira, I., Bento, I. and Bettencourt-Dias, M. (2009). From zero to many: control of centriole number in development and disease. Traffic. 10, 482-498.
(9) Doxsey, S., Zimmerman, W. and Mikule, K. (2005). Centrosome control of the cell cycle. Trends Cell Biol. 15, 303-311.
(10) Erhardt, M., Namba, K. and Hughes, K. T. (2010). Bacterial nanomachines: the flagellum and type III injectisome. Cold Spring Harb Perspect Biol. 11, 1-22.
(11) Field, M. C. and Carrington, M. (2009). The trypanosome flagellar pocket. Nat Rev Microbiol. 7, 775-786.
(12) Fliegauf, M., Benzing, T. and Omran, H. (2007). When cilia go bad: cilia defects and ciliopathies. Nat Rev Mol Cell Biol. 8, 880-893.
(13) Fong, H. K. W., Hurley, J. B., Hopkins, R. S., Miake-Lye, R., Johnson, M. S., Doolittle, R. F. and Simon, M. I. (1986). Repetitive segmental structure of the transducin β subunit: Homology with the CDC4 gene and identification of related mRNAs. Proc Natl Acad Sci U S A. 83, 2162-2166.
(14) Garcia-Higuera, I., Fenoglio, J., Li, Y., Lewis, C., Panchenko, M. P., Reiner, O., Smith, T. F. and Neer, E. J. (1996). Folding of proteins with WD-repeats: comparison of six members of the WD-repeat superfamily of the G protein beta subunit. Biochemistry. 35, 13985-13994.
(15) Hammarton, T. C. (2007). Cell cycle regulation in Trypanosoma brucei. Mol Biochem Parasitol. 153, 1-8.
(16) Hildebrandt, F., Benzing, T. and Katsanis, N. (2011). Ciliopathies. N Engl J Med. 364, 1533-1543.
(17) Hirano, T. (2006). At the heart of the chromosome: SMC proteins in action. Nat Rev Mol Cell Biol. 7, 311-322.
(18) Hirano, T. (2005). SMC proteins and chromosome mechanics: from bacteria to humans. Philos Trans R Soc Lond B Biol Sci. 360, 507-514.
(19) Horowitz, E., Zhang, Z., Jones, B. H., Moss, S. B., Ho, C., Wood, J. R., Wang, X., Sammel, M. D. and Strauss, J. F. 3rd.(2005). Patterns of expression of sperm flagellar genes: early expression of genes encoding axonemal proteins during the spermatogenic cycle and shared features of promoters of genes encoding central apparatus proteins. Mol Hum Reprod. 11, 307-317.
(20) Ibañez-Tallon, I., Heintz, N. and Omran, H. (2003). To beat or not to beat: roles of cilia in development and disease. Hum Mol Genet. 12, 27-35.
(21) Jackman, J. and O’Connor, P. M. (2001). Methods for synchronizing cells at specific stages of the cell cycle. Curr Protoc Cell Bio. 10. 1002-1047.
(22) Kasmyth, K. and Haering, C. H. (2005). The structure and function of SMC and kleisin complexes. Annu Rev Biochem. 74, 595-648.
(23) Kennedy, P. G. (2008). The continuing problem of human African trypanosomiasis (sleeping sickness). Ann Neurol. 64, 116-126.
(24) Kobayashi, T. and Dynlacht, B. D. (2011). Regulating the transition from centriole to basal body. J Cell Biol. 193, 435-444.
(25) Kohl, L. and Gull, K. (1998). Molecular architecture of the trypanosome cytoskeleton. Mol Biochem Parasitol. 93, 1-9.
(26) Lacomble, S., Vaughan, S., Gadelha, C., Morphew, M. K., Mclntosh, J. R. and Gull, K. (2010). Basal body movements orchestrate membrane organelle division and cell morphogenesis in Trypanosoma brucei. J Cell Sci. 123, 2884-2891.
(27) Levine, B., Mizushima, N. and Virgin, H. W. (2011). Autophagy in immunity and inflammation. Nature . 469, 323-335.
(28) Lindemann, C. B. and Lesich, K. A. (2010). Flagellar and ciliary beating: the proven and the possible. J Cell Sci. 123, 519-523.
(29) Lundkvist, G. B., Kristensson, K. and Bentivoqlio, M. (2004). Why trypanosomes cause sleeping sickness. Phisiology. 19, 198-206.
(30) Mardin, B. R., Lange, C., Baxter, J. E., Hardy, T., Scholz, S. R., Fry, A. M. and Schiebel, E. (2010). Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction. Nat Cell Biol. 12, 1166-1178.
(31) Margalit, A., Vlcek, S., Gruenbaum, Y. and Foisner, R. (2005). Breaking and making of the nuclear envelope. J Cell Biochem. 95, 454-465.
(32) Nagarkatti-Gude, D. R., Jaimez, R., Henderson, S. C., Teves, M. E., Zhang, Z. and Strauss, F. J. 3rd. (2011). Spag16, an axoneme central apparatus gene, encodes a male germ cell nuclear speckle protein that regulates SPAG16 mRNA expression. PLoS One. 6, 1-12.
(33) Neer, E. J., Schmidt, C. J., Nambudripad, R. and Smith, T. F. (1994). The ancient regulatory-protein family of WD-repeat proteins. Nature. 371, 297-300.
(34) Neer, E. J. and Clapham, D. E. (1988). Roles of G protein subunits in transmembrane signaling. Nature. 333, 129-134.
(35) Nigg, E. A.. (2002). Centrosome aberrations: cause or consequence of cancer progression. Nat Rev Cancer. 11, 815-825.
(36) Niki, H., Jaffѐ, A., Imamura, R., Ogura, T. and Hiraga, S. (1991). The new gene mukB codes for a 177 kd protein with coiled-coil domains involved in chromosome partitioning of E. coli. EMBO J. 10, 183-193.
(37) Nowak, S. J. and Corces, V. G. (2004). Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation. Trends Genet. 20, 214-220.
(38) Orsi, A., Polson, H. E. and Tooze, S. A. (2010). Membrane trafficking events that partake in autophagy. Curr Opin Cell Biol. 2, 150-156.
(39) Pazour, G. J., Dickert, B. L., Vucica, Y. Seeley, E. S., Rosenbaum, J. L., Witman, G. B. and Cole, D. G. (2000). Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene Tg737, are required for assembly of cilia and flagella. J Cell Bio. 151, 709-718.
(40) Ralston, K. S., Lerner, A. G., Diener, D. R. and Hill, K. L. (2006). Flagellar Motility Contributes to Cytokinesis in Trypanosoma brucei and Is Modulated by an Evolutionarily Conserved Dynein Regulatory System. Eukaryot Cell. 5, 696-711.
(41) Rieder, C. L. (2010). Mitosis in vertebrates: the G2/M and M/A transitions and their associated checkpoints. Chromosome Res. 19, 291-306.
(42) Smith, E. F. and Lefebvre, P. A. (1997). PF20 gene product contains WD repeats and localized to the intermicrotubule bridges in Chlamydomonas flagella. Mol Biol Cell. 8, 455-467.
(43) Stirnimann, C. U., Petsalaki, E., Russell, R. B. and Müller, C. W. (2010). WD40 proteins propel cellular networks. Trends Biochem Sci. 35, 565-574.
(44) Uzbekov, R., Kireyev, I. and Prigent, C. (2002). Centrosome separation: respective role of microtubules and actin filaments. Biol Cell. 94, 275-288.
(45) Vaughan, S. and Dawe, H. R. (2011). Common themes in centriole and centrosome movements. Trends Cell Biol. 21, 57-66.
(46) Yu, L., Gaitatzes, C., Neer, E. and Smith, T. F. (2000). Thirty-plus functional families from a single motif. Protein Sci. 9, 2470-2476.
(47) Zhang, Z., Shen, X., Jones, B. H., Xu, B., Herr, J. C. and Strauss, J. F. 3rd. (2008). Phosphorylation of mouse sperm axoneme central apparatus protein SPAG16L by a testis-specific kinase, TSSK2. Biol Reprod. 79, 75-83.
(48) Zhang, Z., Jones, B. H., Tang, W., Moss, S. B., Wei, Z., Ho, C., Pollack, M., Horowitz, E., Bennett, J., Baker, M. E. and Strauss, J. F. 3rd. (2005). Dissecting the axoneme interactome: the mammalian orthologue of Chlamydomonas PF6 interacts with sperm-associated antigen 6, the mammalian orthologue of Chlamydomonas PF16. Mol Cell Proteomics. 10, 914-923.
(49) Zhang, Z., Kostetskii, I., Moss, S. B., Jones, B. H., Ho, C., Wang, H., Kishida, T., Gerton, G. L., Radice, G. L. and Strauss, J. F. 3rd. (2004). Haploinsuffi- ciency for the murine orthologue of Chlamydomonas PF20 disrupts spermato- genesis. Proc Natl Acad Sci U S A. 101, 12946-12951.